Signaltransduktion

1. Signaltransduktion Molekularbiologie und Genetik: Gene und Genome Wintersemester 2008/2009 Katja Arndt

2. Signaltransduktion Signal�bertragungsweg / Signaltransduktionsweg Prozess, durch den Signal an Zelloberfl�che in spezifische Zellantwort umgesetzt wird, besteht aus mehreren Schritten. Derartige Mechanismen entwickelten sich vermutlich bereits bei den Urformen der Pro- und Eukaryoten und wurden bei den viel sp�ter entstandenen vielzelligen Organismen f�r neue Funktionen abgewandelt. Unsere heutigen Kenntnisse gehen auf die Pionierarbeiten von Earl W. Sutherland zur�ck, der 1971 den Nobelpreis erhielt: �for his discoveries concerning the mechanisms of the action of hormones.� Sutherland untersuchte, wie Adrenalin in Leber- und Skelettmuskelzellen den Abbau von Glykogen anregt.

3. �berblick Einleitung Signaltransduktion, Zell-Kommunikation Rezeptoren Zelloberfl�chen-Rezeptoren: A) Ionenkanal-Rezeptoren B) G-Protein gekoppelte Rezeptoren C) Enzymgekoppelte Rezeptoren Intrazellul�re Rezeptoren Signal�bertragung und Zellantwort Signalwege �ber G-Proteine: A) Adenylatcyclase, cAMP, PKA B) Phospholipase C, DAG, IP3 Signalwege �ber enzymgekoppelte Rezeptoren: A) Ras, MAP-Kinase

4. Signal�bertragungswege Cytokines, Growth Factors and Hormones Cytokines, growth factors (GF), and hormones are all chemical messengers that mediate intercellular communication. The regulation of cellular and nuclear functions by cytokines, growth factors, and peptide or protein hormones is initiated through the activation of cell surface receptors (Rc). All receptors have two main components: 1) a ligand-binding domain that ensures ligand specificity and 2) an effector domain that initiates the generation of the biological response upon ligand binding. The activated receptor may then interact with other cellular components to complete the signal transduction process. Many growth factors bind to receptors that are linked through G-proteins to membrane-bound phospholipase C (PLC). Activation of PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerols (DAG) and D-myo-inositol-1, 4, 5-trisphosphate (IP3). IP3 regulates intracellular Ca2+ by binding to the IP3 receptor on the endoplasmic reticulum (ER) and stimulating Ca2+ release from the ER. Free intracellular Ca2+ can bind to calmodulin, and this Ca2+-calmodulin complex, in the presence of cyclic-AMP (cAMP), activates protein kinase A (PKA) by binding to the regulatory subunit of the enzyme. DAG binds to and activates protein kinase C (PKC). Other hormone receptors may be linked through G-proteins to adenyl cyclase (AC) instead of PLC. Activation of AC increases the cellular levels of cAMP and, in the presence of the Ca2+-calmodulin complex, will activate PKA. Additionally, some growth factor and cytokine receptors are protein tyrosine kinases (PTK) that are directly activated by ligand-receptor interaction. Activation of any of the protein kinases, PKA, PKC, or PTK, catalyzes the phosphorylation of other proteins within the cell. Enzymes that are activated or inhibited by phosphorylation may mediate functional processes within the cell, while others may be one step in a protein kinase cascade that regulates nuclear events. Steroid hormones (i.e. estrogen, glucocorticoids), thyroid hormone, vitamin D3, and retinoids are all small lipophilic molecules that easily penetrate both the cellular and nuclear membranes to enter the nucleus where they bind to their respective receptors that are ligand-dependent transcription factors. These ligand-receptor complexes bind to specific DNA response elements in the promoter region and regulate gene expression. References Luttrell, L.M., et al., G-protein-coupled receptors and their regulation: activation of the MAP kinase signaling pathway by G-protein-coupled receptors. Adv. Second Messenger Phosphoprotein Res., 31, 263-277 (1997). Marshall, C.J., Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell., 80,179-185 (1995). Kumar, R., Thompson, E.B., The structure of the nuclear hormone receptors. Steroids, 64, 310-319 (1999). Cytokines, Growth Factors and Hormones Cytokines, growth factors (GF), and hormones are all chemical messengers that mediate intercellular communication. The regulation of cellular and nuclear functions by cytokines, growth factors, and peptide or protein hormones is initiated through the activation of cell surface receptors (Rc). All receptors have two main components: 1) a ligand-binding domain that ensures ligand specificity and 2) an effector domain that initiates the generation of the biological response upon ligand binding. The activated receptor may then interact with other cellular components to complete the signal transduction process. Many growth factors bind to receptors that are linked through G-proteins to membrane-bound phospholipase C (PLC). Activation of PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerols (DAG) and D-myo-inositol-1, 4, 5-trisphosphate (IP3). IP3 regulates intracellular Ca2+ by binding to the IP3 receptor on the endoplasmic reticulum (ER) and stimulating Ca2+ release from the ER. Free intracellular Ca2+ can bind to calmodulin, and this Ca2+-calmodulin complex, in the presence of cyclic-AMP (cAMP), activates protein kinase A (PKA) by binding to the regulatory subunit of the enzyme. DAG binds to and activates protein kinase C (PKC). Other hormone receptors may be linked through G-proteins to adenyl cyclase (AC) instead of PLC. Activation of AC increases the cellular levels of cAMP and, in the presence of the Ca2+-calmodulin complex, will activate PKA. Additionally, some growth factor and cytokine receptors are protein tyrosine kinases (PTK) that are directly activated by ligand-receptor interaction. Activation of any of the protein kinases, PKA, PKC, or PTK, catalyzes the phosphorylation of other proteins within the cell. Enzymes that are activated or inhibited by phosphorylation may mediate functional processes within the cell, while others may be one step in a protein kinase cascade that regulates nuclear events. Steroid hormones (i.e. estrogen, glucocorticoids), thyroid hormone, vitamin D3, and retinoids are all small lipophilic molecules that easily penetrate both the cellular and nuclear membranes to enter the nucleus where they bind to their respective receptors that are ligand-dependent transcription factors. These ligand-receptor complexes bind to specific DNA response elements in the promoter region and regulate gene expression. References Luttrell, L.M., et al., G-protein-coupled receptors and their regulation: activation of the MAP kinase signaling pathway by G-protein-coupled receptors. Adv. Second Messenger Phosphoprotein Res., 31, 263-277 (1997). Marshall, C.J., Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell., 80,179-185 (1995). Kumar, R., Thompson, E.B., The structure of the nuclear hormone receptors. Steroids, 64, 310-319 (1999).

5. Direkte Zell-Zell Kommunikation Direkte Verbindung des Cytoplasmas benachbarter Zellen in Tieren (Gap junctions) und Pflanzen (Plasmodesmata) ? erlaubt eine direkte Diffusion von im Cytosol gel�sten Signalmolek�len. Gap junction: Austausch nur von kleinen Molek�len, z.B. Ca2+, cAMP, nicht jedoch Makromolek�le wie Proteine, DNA.Gap junction: Austausch nur von kleinen Molek�len, z.B. Ca2+, cAMP, nicht jedoch Makromolek�le wie Proteine, DNA.

6. Direkte Zell-Zell Kommunikation Tierzellen kommunizieren auch �ber sich unmittelbar ber�hrende Oberfl�chenmolek�le. Diese Art der Signal�bertragung ist vor allem in der embryonalen Entwicklung und beim Immunsystem von Bedeutung. Gap junction: Austausch nur von kleinen Molek�len, z.B. Ca2+, cAMP, nicht jedoch Makromolek�le wie Proteine, DNA.Gap junction: Austausch nur von kleinen Molek�len, z.B. Ca2+, cAMP, nicht jedoch Makromolek�le wie Proteine, DNA.

7. Lokaler Signalaustausch Signale von einer einzigen Zelle k�nnen viele Zellen in der n�heren Umgebung ansprechen. Solche lokal wirkende Signale bei Tieren sind Parakrine Faktoren. Autokrine Faktoren wirken auf dieselbe Zelle. Neurotransmitter:Spalt kleiner als 100 nm, Diffusion schneller als eine msec. Hohe Konz., Z.B. Acetylcholinkonz. ca. 5x10^4 M. Neurotransmitter schnell aus dem synaptischen Spalt entfernt => schnelles, pr�zises Signal. Autokrine Signalgebung: Faktoren wirken auf die selbe Zelle, wichtig bei Entwicklung, �Kommunityeffekt�, wird auch von Krebszellen zur Proliferation genutzt.Neurotransmitter:Spalt kleiner als 100 nm, Diffusion schneller als eine msec. Hohe Konz., Z.B. Acetylcholinkonz. ca. 5x10^4 M. Neurotransmitter schnell aus dem synaptischen Spalt entfernt => schnelles, pr�zises Signal. Autokrine Signalgebung: Faktoren wirken auf die selbe Zelle, wichtig bei Entwicklung, �Kommunityeffekt�, wird auch von Krebszellen zur Proliferation genutzt.

8. Lokaler Signalaustausch Eine Nervenzelle produziert ein chemisches Signal (Neurotransmitter), das zu einer einzelnen Zielzelle diffundiert. Ein elektrisches Signal wird �ber die gesamte L�nge der Nervenzelle weitergeleitet und l�st im synaptischen Spalt die Aussch�ttung von Neurotransmittern aus. Ein Signal kann so gezielt �ber weite Distanzen weitergeleitet werden. Schnelle Diffusion (<1 ms) Schnelles pr�zises Signal Neurotransmitter:Spalt kleiner als 100 nm, Diffusion schneller als eine msec. Hohe Konz., Z.B. Acetylcholinkonz. ca. 5x10^4 M. Neurotransmitter schnell aus dem synaptischen Spalt entfernt => schnelles, pr�zises Signal. Autokrine Signalgebung: Faktoren wirken auf die selbe Zelle, wichtig bei Entwicklung, �Kommunityeffekt�, wird auch von Krebszellen zur Proliferation genutzt.Neurotransmitter:Spalt kleiner als 100 nm, Diffusion schneller als eine msec. Hohe Konz., Z.B. Acetylcholinkonz. ca. 5x10^4 M. Neurotransmitter schnell aus dem synaptischen Spalt entfernt => schnelles, pr�zises Signal. Autokrine Signalgebung: Faktoren wirken auf die selbe Zelle, wichtig bei Entwicklung, �Kommunityeffekt�, wird auch von Krebszellen zur Proliferation genutzt.

9. Signalaustausch �ber weite Entfernung Signalaustausch �ber gro�e Entfernung erfolgt bei Tieren und Pflanzen durch Hormone. Das Hormonsystem in Tieren wird endokrines System genannt. Endokrines System ist langsamer (z.B. Signal bewirkt Ver�nderung der Genexpression) Endokrines System langsamer, besonders wenn Signal Ver�nderung der Genexpression bewirkt. Hoch affine Rezeptoren, da Hormone im Blut stark verd�nnt, Konz. meist <10^8 M.Endokrines System langsamer, besonders wenn Signal Ver�nderung der Genexpression bewirkt. Hoch affine Rezeptoren, da Hormone im Blut stark verd�nnt, Konz. meist <10^8 M.

10. �berblick Signaltransduktion



13. Typen von Rezeptoren Rezeptorproteine meist in der Plasmamembran. Signalmolek�le oft wasserl�slich und gro� und daher nicht membrang�ngig. Rezeptor vermittelt Signal von der Zelloberfl�che ins Zellinnere durch Konformations�nderung oder Aggregation. Wichtigste Typen von Membran- rezeptoren auf der Zelloberfl�che: Ionenkanal-Rezeptoren G-Protein gekoppelte Rezeptoren Enzymgekoppelte Rezeptoren Rezeptoren im Zellinneren: Rezeptoren f�r Steroid- und Schilddr�senhormone, NO

14. A) Ionenkanal-Rezeptoren Ligandengesteuerte Ionenkan�le sind Proteinporen in der Plasmamembran

15. Beispiel: Chemische Synapse Neurotransmitter werden von der pr�synaptischen Zelle ausgesch�ttet und binden den Rezeptor des postsynaptischen Ionenkanals. Neurotransmitter werden schnell abgebaut oder von anderen Neuronen aufgenommen, so dass das Signal kurz und pr�zise ist.

16. B) G-Protein gekoppelte Rezeptoren ... wechselwirken mit einem intrazellul�r an der Membran verankertem G-Protein (= GTP bindendes Protein), das das Signal weiterleitet. ... bilden die gr��te Familie der Zelloberfl�chen-Rezeptoren aus (~1000 involviert in Geruchserkennung). ... binden eine Vielzahl von unterschiedlichen Signalmolek�len. ... kommen in verschiedenen Familien vor (z.B. Adrenalin bindet an mind. 9 verschiedene Rezeptoren).

17. Struktur des Rezeptors Three-dimensional structure of rhodopsin, a 7TM receptor taking part in visual signal transduction. As the first 7TM receptor whose structure has been determined, its structure provides a framework for understanding other 7TM receptors. A linked photoreceptor molecule, retinal, is present in the position where, in at least other 7TM receptors, ligands likely bind. Three-dimensional structure of rhodopsin, a 7TM receptor taking part in visual signal transduction. As the first 7TM receptor whose structure has been determined, its structure provides a framework for understanding other 7TM receptors. A linked photoreceptor molecule, retinal, is present in the position where, in at least other 7TM receptors, ligands likely bind.

18. G-Proteine als molekularer Schalter

19. G-Proteine The structure of an inactive G protein. (A) Note that both the a and the ? subunits have covalently attached lipid molecules (red) that help to bind them to the plasma membrane, and the a subunit has GDP bound. (B) The three-dimensional structure of an inactive G protein, based on transducin, the G protein in visual transduction (discussed later). The a subunit contains the GTPase domain and binds to one side of the � subunit, which locks the GTPase domain in an inactive conformation that binds GDP. The ? subunit binds to the opposite side of the � subunit. (B, based on D.G. Lombright et al., Nature 379:311�319, 1996.) The structure of an inactive G protein. (A) Note that both the a and the ? subunits have covalently attached lipid molecules (red) that help to bind them to the plasma membrane, and the a subunit has GDP bound. (B) The three-dimensional structure of an inactive G protein, based on transducin, the G protein in visual transduction (discussed later). The a subunit contains the GTPase domain and binds to one side of the � subunit, which locks the GTPase domain in an inactive conformation that binds GDP. The ? subunit binds to the opposite side of the � subunit. (B, based on D.G. Lombright et al., Nature 379:311�319, 1996.)

20. Aktivierung der G-Proteine Der Rezeptor bleibt aktiv solange ein Signalmolek�l gebunden ist. (A) In the unstimulated state, the receptor and the G protein are both inactive. Although they are shown here as separate entities in the plasma membrane, in some cases, at least, they are associated in a preformed complex. (B) Binding of an extracellular signal to the receptor changes the conformation of the receptor, which in turn alters the conformation of the G protein that is bound to the receptor. (C) The alteration of the a subunit of the G protein allows it to exchange its GDP for GTP. This causes the G protein to break up into two active components�an a subunit and a �? complex, both of which can regulate the activity of target proteins in the plasma membrane. The receptor stays active while the external signal molecule is bound to it, and it can therefore catalyze the activation of many molecules of G protein. (A) In the unstimulated state, the receptor and the G protein are both inactive. Although they are shown here as separate entities in the plasma membrane, in some cases, at least, they are associated in a preformed complex. (B) Binding of an extracellular signal to the receptor changes the conformation of the receptor, which in turn alters the conformation of the G protein that is bound to the receptor. (C) The alteration of the a subunit of the G protein allows it to exchange its GDP for GTP. This causes the G protein to break up into two active components�an a subunit and a �? complex, both of which can regulate the activity of target proteins in the plasma membrane. The receptor stays active while the external signal molecule is bound to it, and it can therefore catalyze the activation of many molecules of G protein.

21. Konformations�nderung der a-Untereinheit

22. Aktivierung von Zielmolek�len The switching off of the G-protein a subunit by the hydrolysis of its bound GTP. After a G-protein a subunit activates its target protein, it shuts itself off by hydrolyzing its bound GTP to GDP. This inactivates the a subunit, which dissociates from the target protein and reassociates with a �? complex to re-form an inactive G protein. Binding to the target protein or to a membrane-bound RGS protein (not shown) usually stimulates the GTPase activity of the a subunit; this stimulation greatly speeds up the inactivation process shown here. The switching off of the G-protein a subunit by the hydrolysis of its bound GTP. After a G-protein a subunit activates its target protein, it shuts itself off by hydrolyzing its bound GTP to GDP. This inactivates the a subunit, which dissociates from the target protein and reassociates with a �? complex to re-form an inactive G protein. Binding to the target protein or to a membrane-bound RGS protein (not shown) usually stimulates the GTPase activity of the a subunit; this stimulation greatly speeds up the inactivation process shown here.

23. Inaktivierung der G-Proteine The switching off of the G-protein a subunit by the hydrolysis of its bound GTP. After a G-protein a subunit activates its target protein, it shuts itself off by hydrolyzing its bound GTP to GDP. This inactivates the a subunit, which dissociates from the target protein and reassociates with a �? complex to re-form an inactive G protein. Binding to the target protein or to a membrane-bound RGS protein (not shown) usually stimulates the GTPase activity of the a subunit; this stimulation greatly speeds up the inactivation process shown here. The switching off of the G-protein a subunit by the hydrolysis of its bound GTP. After a G-protein a subunit activates its target protein, it shuts itself off by hydrolyzing its bound GTP to GDP. This inactivates the a subunit, which dissociates from the target protein and reassociates with a �? complex to re-form an inactive G protein. Binding to the target protein or to a membrane-bound RGS protein (not shown) usually stimulates the GTPase activity of the a subunit; this stimulation greatly speeds up the inactivation process shown here.

24. Diversit�t G-Protein gekoppelter Signalwege Diversity of G Protein-Coupled Receptor Signal Transduction Pathways Receptors coupled to heterotrimeric GTP-binding proteins (G proteins) are integral transmembrane proteins that transduce extracellular signals to the cell interior. G protein-coupled receptors exhibit a common structural motif consisting of seven membrane spanning regions. Receptor occupation promotes interaction between the receptor and the G protein on the interior surface of the membrane. This induces an exchange of GDP for GTP on the G protein ? subunit and dissociation of the ? subunit from the ?? heterodimer. Depending on its isoform, the GTP-? subunit complex mediates intracellular signaling either indirectly by acting on effector molecules such as adenylyl cyclase (AC) or phospholipase C (PLC), or directly by regulating ion channel or kinase function. References Schoneberg, T., et al., Structural basis of G protein-coupled receptor function. Mol. Cell. Endocrinol., 151, 181-193 (1999). LeVine, H., 3rd., Structural features of heterotrimeric G protein-coupled receptors and their modulatory proteins. Mol. Neurobiol., 19, 111-149 (1999). Morris, A.J., et al., Physiological regulation of G protein-linked signaling. Physiol. Rev., 79, 1373-1430 (1999). Diversity of G Protein-Coupled Receptor Signal Transduction Pathways Receptors coupled to heterotrimeric GTP-binding proteins (G proteins) are integral transmembrane proteins that transduce extracellular signals to the cell interior. G protein-coupled receptors exhibit a common structural motif consisting of seven membrane spanning regions. Receptor occupation promotes interaction between the receptor and the G protein on the interior surface of the membrane. This induces an exchange of GDP for GTP on the G protein ? subunit and dissociation of the ? subunit from the ?? heterodimer. Depending on its isoform, the GTP-? subunit complex mediates intracellular signaling either indirectly by acting on effector molecules such as adenylyl cyclase (AC) or phospholipase C (PLC), or directly by regulating ion channel or kinase function. References Schoneberg, T., et al., Structural basis of G protein-coupled receptor function. Mol. Cell. Endocrinol., 151, 181-193 (1999). LeVine, H., 3rd., Structural features of heterotrimeric G protein-coupled receptors and their modulatory proteins. Mol. Neurobiol., 19, 111-149 (1999). Morris, A.J., et al., Physiological regulation of G protein-linked signaling. Physiol. Rev., 79, 1373-1430 (1999).

25. Diversit�t von G-Protein gekoppelten Signalwegen

26. C) Enzymgekoppelte Rezeptoren Bindung von Wachstumsfaktoren sehr geringer Konzentration (nM - pM). Signalantwort in der Regel langsam (Stunden) und �ber mehrere Schritte. Einteilung in sechs Klassen: Rezeptor Tyrosinkinasen (RTK): Phosphorylieren Tyrosine von spezifischen intrazellul�ren Signalproteinen. Tyrosinkinase assoziierte Rezeptoren: Assoziieren mit intrazellul�ren Proteinen, die Tyrosinkinase Aktivit�t besitzen. Rezeptor�hnliche Tyrosinphosphatasen: Entfernen Phosphatgruppen von Tyrosinsn von spezifischen intrazellul�ren Signalproteinen. Rezeptor Serin/Threoninkinasen: Phosphorylieren spezifische Ser oder Thr von assoziierten Genregulationsproteinen. Rezeptor Guanylyl Cyclasen: Katalysieren direkt die Produktion von cGMP im Cytosol. Histinkinase assoziierte Rezeptoren: Aktivieren einen �zwei Komponenten� Signalweg, bei dem die Kinase sich selbst phosphoryliert and dann dieses Phosphat sofort auf ein sekund�res Signalprotein �bertragen wird. Rezeptor�hnliche Tyrosinphosphatasen: �hnlich, weil bislang noch kein Ligand identifiziertRezeptor�hnliche Tyrosinphosphatasen: �hnlich, weil bislang noch kein Ligand identifiziert

27. Rezeptor-Tyrosinkinasen: Subfamilien Transmembranproteine: extrazellul�rer Ligandbindungsstelle eine Transmembranhelix cytosolischer Dom�ne mit Enzymaktivit�t oder direkt assoziiert mit Enzym. Klassifizierung in verschiedene Subfamilien. SIGNALING LIGANDRECEPTORSSOME RESPONSES Epidermal growth factor (EGF)EGF receptorstimulates proliferation of various cell types Insulininsulin receptorstimulates carbohydrate utilization and protein synthesis Insulin-like growth factors (IGF-1 and IGF-2)IGF receptor-1stimulate cell growth and survival Nerve growth factor (NGF)Trk Astimulates survival and growth of some neurons Platelet-derived growth factors (PDGF AA, BB, AB)PDGF receptors (a and �)stimulate survival, growth, and proliferation of various cell types Macrophage-colony-stimulating (M-CSF)M-CSF receptor factorstimulates monocyte/macrophage proliferation and differentiation Fibroblast growth factors (FGF-1 to FGF-24)FGF receptors (FGF-R1-FGF- R4, plus multiple isoforms of each)stimulate proliferation of various cell types; inhibit differentiation of some precursor cells; inductive signals in development Vascular endothelial growth factor (VEGF)VEGF receptorstimulates angiogenesisEphrins (A and B types) Eph receptors (A and B types)stimulate angiogenesis; guide cell and axon migrationSIGNALING LIGANDRECEPTORSSOME RESPONSES Epidermal growth factor (EGF)EGF receptorstimulates proliferation of various cell types Insulininsulin receptorstimulates carbohydrate utilization and protein synthesis Insulin-like growth factors (IGF-1 and IGF-2)IGF receptor-1stimulate cell growth and survival Nerve growth factor (NGF)Trk Astimulates survival and growth of some neurons Platelet-derived growth factors (PDGF AA, BB, AB)PDGF receptors (a and �)stimulate survival, growth, and proliferation of various cell types Macrophage-colony-stimulating (M-CSF)M-CSF receptor factorstimulates monocyte/macrophage proliferation and differentiation Fibroblast growth factors (FGF-1 to FGF-24)FGF receptors (FGF-R1-FGF- R4, plus multiple isoforms of each)stimulate proliferation of various cell types; inhibit differentiation of some precursor cells; inductive signals in development Vascular endothelial growth factor (VEGF)VEGF receptorstimulates angiogenesisEphrins (A and B types) Eph receptors (A and B types)stimulate angiogenesis; guide cell and axon migration

28. Rezeptor-Tyrosinkinasen: Aktivierung Katalysieren die �bertragung einer Phosphat-Gruppe von ATP auf spezifische Tyrosinreste im Rezeptormolek�l (Autophosphorylierung) oder in assoziierten intrazellul�ren Substratproteinen.

29. Beispiele f�r Rezeptor �Crosslinking� Platelet-derived growth factor (PDGF): Kovalent verbundenes Dimer mit zwei Rezeptor-Bindungsstellen. Fibroblast growth factors (FGFs): Monomer, bindet in Klustern an Proteoglykane und kann dar�ber Rezeptor vernetzen. Ephrin: Monomer, membrangebunden, Vernetzung durch Kluster in der Zellmembran.

30. Phosphorylierungskaskade

31. Der Gegenspieler von Proteinkinasen: Protein Phosphatasen Dephosphorylierung von pSer und pThr durch 4 Typen von Serin/Threonin-Phosphoprotein Phosphatasen: Typ I: Dephosphoryliert die meisten Proteine, die durch PKA phosphoryliert wurden. Typ IIA: sehr breite Spezifit�t, hebt viele durch Serin/Threonin-Kinasen verursachte Phosphorylierungen auf. Typ IIB (Calcineurin): wird durch Ca2+ aktiviert, vorwiegend im Gehirn. Typ IIC: nicht mit den anderen Typen verwandt. Phosphatasen (au�er Typ IIC) bestehen aus einer homologen katalytischen Untereinheit und unterschiedlich vielen regulatorischen Untereinheiten.

32. Intrazellul�re-Rezeptoren Some signaling molecules that bind to nuclear receptors. Note that all of them are small and hydrophobic. The active, hydroxylated form of vitamin D3 is shown. Estradiol and testosterone are steroid sex hormones. Some signaling molecules that bind to nuclear receptors. Note that all of them are small and hydrophobic. The active, hydroxylated form of vitamin D3 is shown. Estradiol and testosterone are steroid sex hormones.

33. Membrang�ngige Signalmolek�le Signalmolek�le sind hydrophob und binden an spezifische Tr�gerproteine f�r den Transport in extrazellul�rer Fl�ssigkeit. Beispiele f�r membrang�ngige Signalmolek�le: Steroid Hormone Schilddr�senhormone Retinoide NO

34. Wirkungsweise eines Kern-Rezeptors The nuclear receptor superfamily. All nuclear hormone receptors bind to DNA as either homodimers or heterodimers, but for simplicity we show them as monomers here. (A) The receptors all have a related structure. The short DNA-binding domain in each receptor is shown in green. (B) A receptor protein in its inactive state is bound to inhibitory proteins. Domain-swap experiments suggest that many of the ligand-binding, transcription-activating, and DNA-binding domains in these receptors can function as interchangeable modules. (C) The binding of ligand to the receptor causes the ligand-binding domain of the receptor to clamp shut around the ligand, the inhibitory proteins to dissociate, and coactivator proteins to bind to the receptor's transcription-activating domain, thereby increasing gene transcription. (D) The three-dimensional structure of a ligand-binding domain with (right) and without (left) ligand bound. Note that the bluea helix acts as a lid that snaps shut when the ligand (shown in red) binds, trapping the ligand in place. The nuclear receptor superfamily. All nuclear hormone receptors bind to DNA as either homodimers or heterodimers, but for simplicity we show them as monomers here. (A) The receptors all have a related structure. The short DNA-binding domain in each receptor is shown in green. (B) A receptor protein in its inactive state is bound to inhibitory proteins. Domain-swap experiments suggest that many of the ligand-binding, transcription-activating, and DNA-binding domains in these receptors can function as interchangeable modules. (C) The binding of ligand to the receptor causes the ligand-binding domain of the receptor to clamp shut around the ligand, the inhibitory proteins to dissociate, and coactivator proteins to bind to the receptor's transcription-activating domain, thereby increasing gene transcription. (D) The three-dimensional structure of a ligand-binding domain with (right) and without (left) ligand bound. Note that the bluea helix acts as a lid that snaps shut when the ligand (shown in red) binds, trapping the ligand in place.

35. Wirkungsweise von NO NO reguliert z.B. Kontraktion der glatten Muskulatur: Acetylcholin aus Nervenzellen stimuliert NO Synthese in Endothelzellen. NO diffundiert zu benachbarten Muskelzellen und bewirkt die Bildung von cGMP. Halbwertszeit von NO sehr kurz, 5-10 Sekunden. The role of nitric oxide (NO) in smooth muscle relaxation in a blood vessel wall. Acetylcholine released by nerve terminals in the blood vessel wall activates NO synthase in endothelial cells lining the blood vessel, causing the endothelial cells to produce NO. The NO diffuses out of the endothelial cells and into the underlying smooth muscle cells, where it binds to and activates guanylyl cyclase to produce cyclic GMP. The cyclic GMP triggers a response that causes the smooth muscle cells to relax, enhancing blood flow through the blood vessel. The role of nitric oxide (NO) in smooth muscle relaxation in a blood vessel wall. Acetylcholine released by nerve terminals in the blood vessel wall activates NO synthase in endothelial cells lining the blood vessel, causing the endothelial cells to produce NO. The NO diffuses out of the endothelial cells and into the underlying smooth muscle cells, where it binds to and activates guanylyl cyclase to produce cyclic GMP. The cyclic GMP triggers a response that causes the smooth muscle cells to relax, enhancing blood flow through the blood vessel.

36. �berblick Einleitung Signaltransduktion, Zell-Kommunikation Rezeptoren Zelloberfl�chen-Rezeptoren: A) Ionenkanal-Rezeptoren B) G-Protein gekoppelte Rezeptoren C) Enzymgekoppelte Rezeptoren Intrazellul�re Rezeptoren Signal�bertragung und Zellantwort Signalwege �ber G-Proteine: A) Adenylatcyclase, cAMP, PKA B) Phospholipase C, DAG, IP3 Signalwege �ber enzymgekoppelte Rezeptoren: A) Ras, MAP-Kinase

37. Signal�bertragungswege Relay protein: �bermittelt Signal zur n�chsten Kompenente des Signalweges. Adaptor protein: verbindet Signalmolek�le miteinander ohne selbst an der Signal�bertragung beteiligt zu sein. Amplifier protein: (meist Enzym oder Ionenkanal) vervielf�ltigt Signal durch Produktion einer Vielzahl kleiner Signalmolek�le oder durch Aktivierung von vielen Signalproteinen. Bei vielen solchen Schritten spricht man von Signalkaskade. Transducer protein: konvertiert Signal in andere Form (z.B. Enzym synthetisiert cAMP). Messenger protein: �bermittelt Signal von einem Teil der Zelle zu einem anderen (z.B. vom Cytosol zum Kern). Bifurcation protein: �bermitteln Signal von einem Signalweg zu einem anderen. Integrator protein: vereinigen Signale aus verschiedenen Signalwegen. Latent regulatory protein: wird an der Zelloberfl�che durch aktivierte Rezeptoren stimuliert, wandert in den Zellkern und stimuliert Gentranskription. Different kinds of intracellular signaling proteins along a signaling pathway from a cell-surface receptor to the nucleus. In this example, a series of signaling proteins and small intracellular mediators relay the extracellular signal into the cell, causing a change in gene expression. The signal is amplified, altered (transduced), and distributed en route. Many of the steps can be modulated by other extracellular and intracellular signals, so that the final result of one signal depends on other factors affecting the cell (see Figure 15-8). Ultimately, the signaling pathway activates (or inactivates) target proteins that alter cell behavior. In this example, the target is a gene regulatory protein. Different kinds of intracellular signaling proteins along a signaling pathway from a cell-surface receptor to the nucleus. In this example, a series of signaling proteins and small intracellular mediators relay the extracellular signal into the cell, causing a change in gene expression. The signal is amplified, altered (transduced), and distributed en route. Many of the steps can be modulated by other extracellular and intracellular signals, so that the final result of one signal depends on other factors affecting the cell (see Figure 15-8). Ultimately, the signaling pathway activates (or inactivates) target proteins that alter cell behavior. In this example, the target is a gene regulatory protein.

38. Signal�bertragungswege Modulator protein: modifiziert die Aktivit�t von Signalmolek�len und reguliert damit die St�rke des Signals. Anchoring protein: h�lt spezifische Signalmolek�le an einer bestimmten Stelle in der Zelle durch Anbindung an eine Membran oder das Cytoskelett. Scaffold protein: ist ein Adaptor und/oder Anchoring protein, das verschiedene Signalproteine in einem funktionalen Komplex vereinigt und h�ufig an eine bestimmte Stelle bindet. Different kinds of intracellular signaling proteins along a signaling pathway from a cell-surface receptor to the nucleus. In this example, a series of signaling proteins and small intracellular mediators relay the extracellular signal into the cell, causing a change in gene expression. The signal is amplified, altered (transduced), and distributed en route. Many of the steps can be modulated by other extracellular and intracellular signals, so that the final result of one signal depends on other factors affecting the cell (see Figure 15-8). Ultimately, the signaling pathway activates (or inactivates) target proteins that alter cell behavior. In this example, the target is a gene regulatory protein. Different kinds of intracellular signaling proteins along a signaling pathway from a cell-surface receptor to the nucleus. In this example, a series of signaling proteins and small intracellular mediators relay the extracellular signal into the cell, causing a change in gene expression. The signal is amplified, altered (transduced), and distributed en route. Many of the steps can be modulated by other extracellular and intracellular signals, so that the final result of one signal depends on other factors affecting the cell (see Figure 15-8). Ultimately, the signaling pathway activates (or inactivates) target proteins that alter cell behavior. In this example, the target is a gene regulatory protein.

39. Zwei Hauptwege der Signal�bertragung

40. Signal�bertragungswege Cytokines, Growth Factors and Hormones Cytokines, growth factors (GF), and hormones are all chemical messengers that mediate intercellular communication. The regulation of cellular and nuclear functions by cytokines, growth factors, and peptide or protein hormones is initiated through the activation of cell surface receptors (Rc). All receptors have two main components: 1) a ligand-binding domain that ensures ligand specificity and 2) an effector domain that initiates the generation of the biological response upon ligand binding. The activated receptor may then interact with other cellular components to complete the signal transduction process. Many growth factors bind to receptors that are linked through G-proteins to membrane-bound phospholipase C (PLC). Activation of PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerols (DAG) and D-myo-inositol-1, 4, 5-trisphosphate (IP3). IP3 regulates intracellular Ca2+ by binding to the IP3 receptor on the endoplasmic reticulum (ER) and stimulating Ca2+ release from the ER. Free intracellular Ca2+ can bind to calmodulin, and this Ca2+-calmodulin complex, in the presence of cyclic-AMP (cAMP), activates protein kinase A (PKA) by binding to the regulatory subunit of the enzyme. DAG binds to and activates protein kinase C (PKC). Other hormone receptors may be linked through G-proteins to adenyl cyclase (AC) instead of PLC. Activation of AC increases the cellular levels of cAMP and, in the presence of the Ca2+-calmodulin complex, will activate PKA. Additionally, some growth factor and cytokine receptors are protein tyrosine kinases (PTK) that are directly activated by ligand-receptor interaction. Activation of any of the protein kinases, PKA, PKC, or PTK, catalyzes the phosphorylation of other proteins within the cell. Enzymes that are activated or inhibited by phosphorylation may mediate functional processes within the cell, while others may be one step in a protein kinase cascade that regulates nuclear events. Steroid hormones (i.e. estrogen, glucocorticoids), thyroid hormone, vitamin D3, and retinoids are all small lipophilic molecules that easily penetrate both the cellular and nuclear membranes to enter the nucleus where they bind to their respective receptors that are ligand-dependent transcription factors. These ligand-receptor complexes bind to specific DNA response elements in the promoter region and regulate gene expression. References Luttrell, L.M., et al., G-protein-coupled receptors and their regulation: activation of the MAP kinase signaling pathway by G-protein-coupled receptors. Adv. Second Messenger Phosphoprotein Res., 31, 263-277 (1997). Marshall, C.J., Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell., 80,179-185 (1995). Kumar, R., Thompson, E.B., The structure of the nuclear hormone receptors. Steroids, 64, 310-319 (1999). Cytokines, Growth Factors and Hormones Cytokines, growth factors (GF), and hormones are all chemical messengers that mediate intercellular communication. The regulation of cellular and nuclear functions by cytokines, growth factors, and peptide or protein hormones is initiated through the activation of cell surface receptors (Rc). All receptors have two main components: 1) a ligand-binding domain that ensures ligand specificity and 2) an effector domain that initiates the generation of the biological response upon ligand binding. The activated receptor may then interact with other cellular components to complete the signal transduction process. Many growth factors bind to receptors that are linked through G-proteins to membrane-bound phospholipase C (PLC). Activation of PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to form diacylglycerols (DAG) and D-myo-inositol-1, 4, 5-trisphosphate (IP3). IP3 regulates intracellular Ca2+ by binding to the IP3 receptor on the endoplasmic reticulum (ER) and stimulating Ca2+ release from the ER. Free intracellular Ca2+ can bind to calmodulin, and this Ca2+-calmodulin complex, in the presence of cyclic-AMP (cAMP), activates protein kinase A (PKA) by binding to the regulatory subunit of the enzyme. DAG binds to and activates protein kinase C (PKC). Other hormone receptors may be linked through G-proteins to adenyl cyclase (AC) instead of PLC. Activation of AC increases the cellular levels of cAMP and, in the presence of the Ca2+-calmodulin complex, will activate PKA. Additionally, some growth factor and cytokine receptors are protein tyrosine kinases (PTK) that are directly activated by ligand-receptor interaction. Activation of any of the protein kinases, PKA, PKC, or PTK, catalyzes the phosphorylation of other proteins within the cell. Enzymes that are activated or inhibited by phosphorylation may mediate functional processes within the cell, while others may be one step in a protein kinase cascade that regulates nuclear events. Steroid hormones (i.e. estrogen, glucocorticoids), thyroid hormone, vitamin D3, and retinoids are all small lipophilic molecules that easily penetrate both the cellular and nuclear membranes to enter the nucleus where they bind to their respective receptors that are ligand-dependent transcription factors. These ligand-receptor complexes bind to specific DNA response elements in the promoter region and regulate gene expression. References Luttrell, L.M., et al., G-protein-coupled receptors and their regulation: activation of the MAP kinase signaling pathway by G-protein-coupled receptors. Adv. Second Messenger Phosphoprotein Res., 31, 263-277 (1997). Marshall, C.J., Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell., 80,179-185 (1995). Kumar, R., Thompson, E.B., The structure of the nuclear hormone receptors. Steroids, 64, 310-319 (1999).

41. G-Proteine regulieren Adenylatcyclase

42. Sekund�re Botenstoffe Nicht alle Komponenten eines Signalweges sind Proteine. Sekund�re Botenstoffe (Second Messenger) = kleine, wasserl�sliche Molek�le oder Ionen. (Prim�rer Botenstoff = von au�en kommendes Signal.) Leichte Ausbreitung durch Diffusion erm�glicht die Vermittlung des Signals z.B. von der Zellmembran ins Zellinnere. Die h�ufigsten Sekund�ren Botenstoffe sind: zyklisches AMP (cAMP) Calciumionen (Ca2+) Diacylglycerin (DAG) Inositoltriphosphat (IP3)

43. Bildung von cAMP durch Adenylatcyclase cAMP spielt bei vielen G-Protein vermittelten Signalwegen eine Rolle.

44. Stimulatorische G-Proteine (Gs): aktivieren Adenylat-Cyclase Stimulatorische u. Inhibitorische G-Proteine PGE1 = prostaglandin E1PGE1 = prostaglandin E1

45. Cholera und Pertussis Cholera: BakteriumVibrio cholerae Cholera Toxin ist ein Enzym, das ADP-Ribose von NAD+ auf die a-Untereinheit eines Gs-Proteins �bertr�gt. Dies verhindert die Hydrolyse von gebundenem GTP (G-Protein immer �an�). Aktiviertes G-Protein stimuliert die Adenylat-Cyclase, erh�ht cAMP Konzentration. Erh�hter cAMP Spiegel in den Epithelzellen des Darms bewirkt starken Einstrom von Cl� und Wasser in den Darm, was starken Durchfall ausl�st. Pertussis (Keuchhusten): Bakterium Bordetella pertussis Pertussis Toxin katalysiert die �bertragung von ADP-Ribose auf die a-Untereinheit eines Gi-Proteins. Dies verhindert die Interaktion der a-Untereinheit mit dem Rezeptor, so dass GDP nicht durch GTP ausgetauscht werden kann (G-Protein immer �aus�).

46. Wirkung von cAMP cAMP aktiviert cAMP-abh�ngige Proteinkinase (PKA). cAMP kann auch direkt auf spezielle Ionenkan�le wirken. An increase in cyclic AMP in response to an extracellular signal. This nerve cell in culture is responding to the neurotransmitter serotonin, which acts through a G-protein-linked receptor to cause a rapid rise in the intracellular concentration of cyclic AMP. To monitor the cyclic AMP level, the cell has been loaded with a fluorescent protein that changes its fluorescence when it binds cyclic AMP. Blue indicates a low level of cyclic AMP, yellow an intermediate level, and red a high level. (A) In the resting cell, the cyclic AMP level is about 5 � 10-8 M. (B) Twenty seconds after the addition of serotonin to the culture medium, the intracellular level of cyclic AMP has increased to more than 10-6 M, an increase of more than twentyfold. (From Brian J. Bacskai et al., Science 260:222 226, 1993. � AAAS.) An increase in cyclic AMP in response to an extracellular signal. This nerve cell in culture is responding to the neurotransmitter serotonin, which acts through a G-protein-linked receptor to cause a rapid rise in the intracellular concentration of cyclic AMP. To monitor the cyclic AMP level, the cell has been loaded with a fluorescent protein that changes its fluorescence when it binds cyclic AMP. Blue indicates a low level of cyclic AMP, yellow an intermediate level, and red a high level. (A) In the resting cell, the cyclic AMP level is about 5 � 10-8 M. (B) Twenty seconds after the addition of serotonin to the culture medium, the intracellular level of cyclic AMP has increased to more than 10-6 M, an increase of more than twentyfold. (From Brian J. Bacskai et al., Science 260:222 226, 1993. � AAAS.)

47. cAMP abh�ngige Proteinkinase (PKA) cAMP abh�ngige Proteinkinase (PKA) ist ein Komplex aus zwei katalytischen und zwei regulatorischen Untereinheiten. cAMP aktiviert katalytische Untereinheit katalytische Untereinheit wandert in Kern, aktiviert CREB (=CRE-binding protein). CREB bindet an CRE (cAMP responsive element), aktiviert Gentranskription.

48. Signalkaskaden verst�rken das Signal Beispiel: Glykogenabbau


50. G-Proteine regulieren Phospholipase C-Aktivit�t

51. Phopholipase C bildet zwei sekund�re Botenstoffe (DAG, IP3)

52. Wirkungsweise von IP3 IP3 diffundiert von der Membran durch das Cytosol zum ER und �ffnet Ca2+-Kan�le.

53. Phospholipase C G-Proteine k�nnen membrangebundene Phospholipase C � aktivieren. Modular Structure of Phospholipase C. The domain structures of three isoforms of phospholipase C reveal similarities and differences among the isoforms. Only the b isoform, with the G-protein-binding domain, can be stimulated directly by G proteins. For phospholipase Cg, the insertion of two SH2 (Src homology 2) domains and one SH3 (Src homology 3) domain splits the catalytic domain and a PH domain into two parts. Phospholipase C Acts at the Membrane Surface. The PH and C2 domains of phospholipase help to position the enzyme's catalytic site for ready access to the phosphodiester bond of the membrane lipid substrate, PIP2. Modular Structure of Phospholipase C. The domain structures of three isoforms of phospholipase C reveal similarities and differences among the isoforms. Only the b isoform, with the G-protein-binding domain, can be stimulated directly by G proteins. For phospholipase Cg, the insertion of two SH2 (Src homology 2) domains and one SH3 (Src homology 3) domain splits the catalytic domain and a PH domain into two parts. Phospholipase C Acts at the Membrane Surface. The PH and C2 domains of phospholipase help to position the enzyme's catalytic site for ready access to the phosphodiester bond of the membrane lipid substrate, PIP2.

54. Protein Interaktionsdom�nen

55. Wirkungsweise von DAG Diacylglycerin (DAG) In der Membran verankert. Zwei potentielle Signalwege: Abspaltung von Arachidons�ure: wirkt direkt als Signalgeber. wird f�r die Synthese von Eicosanoiden verwendet (z.B. Prostaglandin). DAG aktiviert Serin/Threonin-Proteinkinase C (PKC)


57. Signalprotein Ras Geh�rt zur Ras-Superfamilie monomerer GTPasen. Aktiviert durch Guanine nucleotide exchange factors (GEF): Austausch von GDP zu GTP (z.B. Sos). Inaktiviert durch GTPase-activating proteins (GAP): beschleunigen Katalyse von gebundenem GTP. Adaptorprotein (z.B. Grb-2) stellt Verbindung zwischen Rezeptor-Tyrosinkinase und Ras her: Grb-2 bindet �ber SH2-Dom�ne an pTyr von RTK und �ber SH3-Dom�ne an Pro-reiche Regionen in GEF Sos. Structure of Grb-2, an Adaptor Protein. Grb-2 consists of two SH3 domains and a central SH2 domain. The SH2 domain binds to phosphotyrosine resides on an activated receptor while the SH3 domains bind proline-rich regions on other proteins such as Sos. Structure of Grb-2, an Adaptor Protein. Grb-2 consists of two SH3 domains and a central SH2 domain. The SH2 domain binds to phosphotyrosine resides on an activated receptor while the SH3 domains bind proline-rich regions on other proteins such as Sos.

58. Ras aktiviert MAP-Kinase Phosphorylierungskaskade Kurzlebige Signale (RTK, Ras) m�ssen in l�ngerlebige Signale umgewandelt werden. Aktiviertes Ras triggert dies durch Aktivierung verschiedener Ser-/Thr-Kinasen. 3 Kern-Module in der Phosphorylierungskaskade: MAP (mitogen-activated protein) Kinase wird spezifisch durch MAP-Kinase-Kinase aktiviert (Phosphorylierung von Ser und Tyr). MAP-Kinase-Kinase wird durch MAP-Kinase-Kinase-Kinase aktiviert. MAP-Kinase-Kinase-Kinase wird durch Ras aktiviert. 30% aller menschlicher Tumore haben hyperaktive ras Mutation. The MAP-kinase serine/threonine phosphorylation pathway activated by Ras. Multiple such pathways involving structurally and functionally related proteins operate in all eucaryotes, each coupling an extracellular stimulus to a variety of cell outputs. The pathway activated by Ras begins with a MAP-kinase-kinase-kinase called Raf, which activates the MAP-kinase-kinase Mek, which then activates the MAP-kinase called Erk. Erk in turn phosphorylates a variety of downstream proteins, including other kinases, as well as gene regulatory proteins in the nucleus. The resulting changes in gene expression and protein activity cause complex changes in cell behavior. The MAP-kinase serine/threonine phosphorylation pathway activated by Ras. Multiple such pathways involving structurally and functionally related proteins operate in all eucaryotes, each coupling an extracellular stimulus to a variety of cell outputs. The pathway activated by Ras begins with a MAP-kinase-kinase-kinase called Raf, which activates the MAP-kinase-kinase Mek, which then activates the MAP-kinase called Erk. Erk in turn phosphorylates a variety of downstream proteins, including other kinases, as well as gene regulatory proteins in the nucleus. The resulting changes in gene expression and protein activity cause complex changes in cell behavior.

59. Verschiedene MAP-Kinase Wege Koexistenz verschiedener MAP-Kinase Wege durch Bindung an Scaffold-Proteine The Mitogen-activated Protein Kinase (MAPK) Cascades Several MAPK cascades have been identified in mammalian cells, including the extracellular signal-related kinase pathways (ERK1/2, ERK5) and the stress activated kinase pathways (JNK/SAPK, p38 MAPK). These pathways are linked to many G protein-linked cell surface receptors and receptor tyrosine kinases. Thus, most cytokines, growth factors, hormones, and neurotransmitters can selectively activate these cascades via receptor activation of intracellular second messengers. All MAPK pathways operate through sequential phosphorylation events to phosphorylate transcription factors and regulate gene expression. They can also phosphorylate cytosolic targets to regulate intracellular events. These cascades are implicated in the regulation of cellular proliferation, differentiation, development, cell cycle, and transmission of oncogenic signals. Courtesy of Rony Seger, Ph.D., Dept. Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel. References: Lowes, V.L., et al., Integration of signals from receptor tyrosine kinases and G protein-coupled receptors. Neurosignals, 11, 5-19 (2002). Tamura, S., et al., Regulation of stress-activated protein kinase signaling pathways by protein phosphatases. Eur. J. Biochem., 269, 1060-1066 (2002). Seger, R., and Krebs, E.G., The MAPK signaling cascade. FASEB J., 9, 726-735 (1995). Koexistenz verschiedener MAP-Kinase Wege durch Bindung an Scaffold-Proteine The Mitogen-activated Protein Kinase (MAPK) Cascades Several MAPK cascades have been identified in mammalian cells, including the extracellular signal-related kinase pathways (ERK1/2, ERK5) and the stress activated kinase pathways (JNK/SAPK, p38 MAPK). These pathways are linked to many G protein-linked cell surface receptors and receptor tyrosine kinases. Thus, most cytokines, growth factors, hormones, and neurotransmitters can selectively activate these cascades via receptor activation of intracellular second messengers. All MAPK pathways operate through sequential phosphorylation events to phosphorylate transcription factors and regulate gene expression. They can also phosphorylate cytosolic targets to regulate intracellular events. These cascades are implicated in the regulation of cellular proliferation, differentiation, development, cell cycle, and transmission of oncogenic signals. Courtesy of Rony Seger, Ph.D., Dept. Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel. References: Lowes, V.L., et al., Integration of signals from receptor tyrosine kinases and G protein-coupled receptors. Neurosignals, 11, 5-19 (2002). Tamura, S., et al., Regulation of stress-activated protein kinase signaling pathways by protein phosphatases. Eur. J. Biochem., 269, 1060-1066 (2002). Seger, R., and Krebs, E.G., The MAPK signaling cascade. FASEB J., 9, 726-735 (1995).

60. Literatur B�cher:

61. Weitere Beispiele

62. Ras aktiviert PI3-Kinase PI (phosphatidylinositol)-3-Kinase Signalweg bewirkt Zellwachstum. PI3-Kinase phosphoryliert Inositolring an Position 3. Verschiedene Typen von PI3-Kinasen. Intrazellul�re Signalmolek�le binden �ber PH-Dom�ne an PI(3,4)P2 und PI(3,4,5)P3. The generation of inositol phospholipid docking sites by PI 3-kinase. PI 3-kinase phosphorylates the inositol ring on carbon atom 3 to generate the inositol phospholipids shown at the bottom of the figure; the two lipids shown in red can serve as docking sites for signaling proteins with PH domains. The phosphorylations indicated by the green arrows are catalyzed by other inositol phospholipid kinases. As discussed earlier, phospholipase C (PLC-b or PLC-g) can cleave PI(4,5)P2 to produce the two small signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate (IP3). The generation of inositol phospholipid docking sites by PI 3-kinase. PI 3-kinase phosphorylates the inositol ring on carbon atom 3 to generate the inositol phospholipids shown at the bottom of the figure; the two lipids shown in red can serve as docking sites for signaling proteins with PH domains. The phosphorylations indicated by the green arrows are catalyzed by other inositol phospholipid kinases. As discussed earlier, phospholipase C (PLC-b or PLC-g) can cleave PI(4,5)P2 to produce the two small signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate (IP3).

63. Beispiel eines PI3-Kinase Signalweges PTEN dephosphoryliert PIP3 (Umkehrreaktion zu PI3-Kinase). PTEN ist ein Tumorsuppressor Gen. Viele Mutationen in Krebszellen befinden sich in dem katalytischen Zentrum von PTEN. Phosphatidylinositol-dependent protein kinase (PDK-1) und Protein-Kinase B (PKB oder Akt) binden PIP3 an der Membran. PDK-1 aktiviert PKB/Akt durch phosphorylierung. Aktiviertes PDB/Akt wandert ins Cytosol und phsophoryliert verschiedene Zielproteine. PTEN Pathway PTEN is a tumor suppressor gene that is able to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5-P3), the product of phosphatidyl inositol 3-kinase (PIK). Many of the mutations that have arisen in cancerous cells have been mapped to the phosphatase catalytic domain of PTEN. Data suggests that the phosphatase activity of PTEN is essential for its function as a tumor suppressor. The activation of Akt/PKB is regulated by the phosphorylation of Akt on Thr308 and Ser473 by phosphoinositide-dependent kinase (PDK) and integrin-linked kinase (ILK), respectively. Inactivation of PTEN allows constitutive and unregulated activation of the Akt/PKB signaling pathway. In addition to regulating the Akt/PKB signaling pathway, PTEN also inhibits growth factor (GF)-induced Shc phosphorylation and suppresses the MAP kinase signaling pathway. PTEN interacts directly with FAK and is able to dephosphorylate activated FAK. PTEN-induced down-regulation of p130CAS through FAK results in inhibition of cell migration and spreading. References Simpson, L., and Parsons, R., PTEN: life as a tumor suppressor. Exp. Cell Res., 264, 29-41 (2001). Besson, A., et al., PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis. Eur. J. Biochem., 263, 605-611 (1999). PTEN Pathway PTEN is a tumor suppressor gene that is able to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate (PI-3,4,5-P3), the product of phosphatidyl inositol 3-kinase (PIK). Many of the mutations that have arisen in cancerous cells have been mapped to the phosphatase catalytic domain of PTEN. Data suggests that the phosphatase activity of PTEN is essential for its function as a tumor suppressor. The activation of Akt/PKB is regulated by the phosphorylation of Akt on Thr308 and Ser473 by phosphoinositide-dependent kinase (PDK) and integrin-linked kinase (ILK), respectively. Inactivation of PTEN allows constitutive and unregulated activation of the Akt/PKB signaling pathway. In addition to regulating the Akt/PKB signaling pathway, PTEN also inhibits growth factor (GF)-induced Shc phosphorylation and suppresses the MAP kinase signaling pathway. PTEN interacts directly with FAK and is able to dephosphorylate activated FAK. PTEN-induced down-regulation of p130CAS through FAK results in inhibition of cell migration and spreading. References Simpson, L., and Parsons, R., PTEN: life as a tumor suppressor. Exp. Cell Res., 264, 29-41 (2001). Besson, A., et al., PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis. Eur. J. Biochem., 263, 605-611 (1999).

64. Src im T-Zellrezeptor Signalweg Viele Zelloberfl�chen-Rezeptoren besitzen keine Tyrosinkinase-Dom�ne sondern sind mit einer cytoplasmatischen Tyrosinkinase assoziiert, die Zielproteine und Rezeptor phosphoryliert. Gr��te Familie der cytoplasmatischen Tyrosinkinasen: Src Familie (Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, Blk). Src reguliert Zellwachstum (Proto-Oncogen). Src Structure. (A) Cellular Src includes an SH3 domain, an SH2 domain, a protein kinase domain, and a carboxyl-terminal tail that includes a key tyrosine residue. (B) Structure of c-Src in an inactivated form with the key tyrosine residue phosphorylated. The phosphotyrosine residue is bound in the SH2 domain; the linker between the SH2 domain and the protein kinase domain is bound by the SH3 domain. These interactions hold the kinase domain in an inactive conformation. T Cell Receptor (TCR) Signaling One of the first steps in the generation of the immune response is the recognition by T lymphocytes of peptide fragments (antigens) derived from foreign pathogens that are presented on the surface of antigen presenting cells (APC). This event is mediated by the T cell receptor (TCR), that transduces these extracellular signals by initiating a wide array of intracellular signaling pathways. One of the first biochemical events following TCR activation is the activation of Src family tyrosine kinases (p56lck) that, in turn, phosphorylate phospholipase C?1 (PLC ?1). Activation of PLC ?1 leads to hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), generating diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates protein kinase C (PKC) that, in turn, phorphorylates Ras, a GTPase that activates Raf leading to recruitment of the MAP kinase cascade. IP3 releases calcium from its intracellular stores in the endoplasmic reticulum (ER). The Ca2+ binds to calmodulin that, in turn, activates calcineurin, a Ca2+/calmodulin dependent protein phosphatase. NFAT, a transcriptional regulator of interleukin-2 (IL-2) gene expression, is a direct target of calcineurin. Calcineurin dephosphorylates the cytosolic component of NFAT, NFATc, which migrates to the nucleus and induces transcription of the IL-2 gene. References Samelson, L.E., et al., Signal transduction mediated by the T-cell antigen receptor. Ann. NY Acad. Sci., 7, 157-172 (1995). Hama, N., et al., Calcium/calmodulin-dependent protein kinase II down regulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells. J. Exp. Med., 181, 1217-1222 (1995). Germain, R.N., and Stefanova, I., The dynamics of T cell receptor signaling: complex orchestration and the key roles of tempo and cooperation. Annu. Rev. Immunol., 17, 467-522 (1999). Src Structure. (A) Cellular Src includes an SH3 domain, an SH2 domain, a protein kinase domain, and a carboxyl-terminal tail that includes a key tyrosine residue. (B) Structure of c-Src in an inactivated form with the key tyrosine residue phosphorylated. The phosphotyrosine residue is bound in the SH2 domain; the linker between the SH2 domain and the protein kinase domain is bound by the SH3 domain. These interactions hold the kinase domain in an inactive conformation. T Cell Receptor (TCR) Signaling One of the first steps in the generation of the immune response is the recognition by T lymphocytes of peptide fragments (antigens) derived from foreign pathogens that are presented on the surface of antigen presenting cells (APC). This event is mediated by the T cell receptor (TCR), that transduces these extracellular signals by initiating a wide array of intracellular signaling pathways. One of the first biochemical events following TCR activation is the activation of Src family tyrosine kinases (p56lck) that, in turn, phosphorylate phospholipase C?1 (PLC ?1). Activation of PLC ?1 leads to hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), generating diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates protein kinase C (PKC) that, in turn, phorphorylates Ras, a GTPase that activates Raf leading to recruitment of the MAP kinase cascade. IP3 releases calcium from its intracellular stores in the endoplasmic reticulum (ER). The Ca2+ binds to calmodulin that, in turn, activates calcineurin, a Ca2+/calmodulin dependent protein phosphatase. NFAT, a transcriptional regulator of interleukin-2 (IL-2) gene expression, is a direct target of calcineurin. Calcineurin dephosphorylates the cytosolic component of NFAT, NFATc, which migrates to the nucleus and induces transcription of the IL-2 gene. References Samelson, L.E., et al., Signal transduction mediated by the T-cell antigen receptor. Ann. NY Acad. Sci., 7, 157-172 (1995). Hama, N., et al., Calcium/calmodulin-dependent protein kinase II down regulates both calcineurin and protein kinase C-mediated pathways for cytokine gene transcription in human T cells. J. Exp. Med., 181, 1217-1222 (1995). Germain, R.N., and Stefanova, I., The dynamics of T cell receptor signaling: complex orchestration and the key roles of tempo and cooperation. Annu. Rev. Immunol., 17, 467-522 (1999).

65. Jak-STAT Signalweg Cytokin-Rezeptoren sind mit cytoplasmatischer Tyrosinkinase der Jak (Janus kinase) Familie (jak1, jak2, jak3, tyk2) assoziiert. The JAK/STAT Signaling Pathway A wide variety of extracellular signals activate the STAT (signal transducers and activators of transcription) class of transcription factors. Many cytokines, lymphokines, and growth factors signal through a related superfamily of cell surface receptor tyrosine kinases that are associated with and activate Janus kinases (JAKs). Ligand-induced dimerization of the receptor induces the reciprocal tyrosine phosphorylation of the associated JAKs, which, in turn, phosphorylates tyrosine residues on the cytoplasmic tail of the receptor. These phosphorylated tyrosines serve as docking sites for the Src Homology-2 (SH-2) domain of the STAT protein, and JAK catalyzes the tyrosine phosphorylation of the receptor-bound STAT. Phosphorylation of STAT at a conserved tyrosine residue induces SH-2-mediated homo- or heterodimerization, followed by translocation of the STAT dimer to the nucleus. STAT dimers bind to specific DNA response elements in the promoter region of target genes to activate gene expression. APS (adaptor molecule containing pleckstrin homology and SH-2 domains) can inhibit the JAK- STAT pathway by binding to the cytoplasmic domain of the receptor where it is phosphorylated (activated) by JAK. Activated APS binds to c-Cbl and blocks STAT activation. References Wakioka, T., et al., APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl. Leukemia, 13, 760-767 (1999). Schindler, C., Cytokines and JAK-STAT signaling. Exp. Cell Res., 253, 7-14 (1999). The JAK/STAT Signaling Pathway A wide variety of extracellular signals activate the STAT (signal transducers and activators of transcription) class of transcription factors. Many cytokines, lymphokines, and growth factors signal through a related superfamily of cell surface receptor tyrosine kinases that are associated with and activate Janus kinases (JAKs). Ligand-induced dimerization of the receptor induces the reciprocal tyrosine phosphorylation of the associated JAKs, which, in turn, phosphorylates tyrosine residues on the cytoplasmic tail of the receptor. These phosphorylated tyrosines serve as docking sites for the Src Homology-2 (SH-2) domain of the STAT protein, and JAK catalyzes the tyrosine phosphorylation of the receptor-bound STAT. Phosphorylation of STAT at a conserved tyrosine residue induces SH-2-mediated homo- or heterodimerization, followed by translocation of the STAT dimer to the nucleus. STAT dimers bind to specific DNA response elements in the promoter region of target genes to activate gene expression. APS (adaptor molecule containing pleckstrin homology and SH-2 domains) can inhibit the JAK- STAT pathway by binding to the cytoplasmic domain of the receptor where it is phosphorylated (activated) by JAK. Activated APS binds to c-Cbl and blocks STAT activation. References Wakioka, T., et al., APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl. Leukemia, 13, 760-767 (1999). Schindler, C., Cytokines and JAK-STAT signaling. Exp. Cell Res., 253, 7-14 (1999).

66. Beispiele von Jak-STAT Signalwegen Some Signaling Proteins That Act Through Cytokine Receptors and the Jak-STAT Signaling PathwaySome Signaling Proteins That Act Through Cytokine Receptors and the Jak-STAT Signaling Pathway

67. TGF-� Signalweg Transforming growth factor-� (TGF-�) Superfamilie besteht aus vielen strukturell verwandten sekretierten dimeren Proteinen. TGF-� aktiviert Rezeptor Ser/Thr-Kinasen Typ I und II. Typ I Rezeptor bindet und phosphoryliert Genregulationsproteine der Smad Familie (Smad1, Smad2, Smad3, Smad5, Smad8). Rezeptoraktiviertes Smad bindet Smad4, wandert in den Kern, bindet mit anderen genregulatorischen Proteinen an spezifische DNA Stellen und aktiviert Zielgene. Regulation: Inhibitorische Smad (Smad6, Smad7): binden an aktivierten TypI-Rezeptor. Interferon ? induziert �ber aktivierten Jak-STAT Weg Produktion von Smad7. Signaling Pathway of TGF-? TGF-? regulates growth and proliferation of cells, blocking the growth of many different cell types. The TGF-? receptor includes Type I and Type II subunits that are serine-threonine kinases that signal through the SMAD family of proteins. Binding of transforming growth factor � (TGF-?) to its cell surface receptor Type II leads to the phosphorylation of the Type I receptor by Type II. The Type I receptor is then able to phosphorylate and activate the Smad2 protein. Smad2, in combination with Smad4, is translocated to the nucleus where the activated Smad complex recruits other transcription factors (TF) that together activate the expression of target genes that mediate the biological effects of TGF-?. Some of the activated target genes stimulate tumorigenesis, while others suppress tumorigenesis. References Kawabata, M., and Miyazono, K., Signal transduction of the TGF-? superfamily by Smad proteins. J. Biochem. (Tokyo), 125, 9-16 (1999). Wrana, J.L., TGF-? receptors and signalling mechanisms. Miner. Electrolyte Metab., 24, 120-130 (1998). Markowitz, S.D., and Roberts, A.B., Tumor suppressor activity of the TGF-? pathway in human cancers. Cytokine Growth Factor Rev., 7, 93-102 (1996). Signaling Pathway of TGF-? TGF-? regulates growth and proliferation of cells, blocking the growth of many different cell types. The TGF-? receptor includes Type I and Type II subunits that are serine-threonine kinases that signal through the SMAD family of proteins. Binding of transforming growth factor � (TGF-?) to its cell surface receptor Type II leads to the phosphorylation of the Type I receptor by Type II. The Type I receptor is then able to phosphorylate and activate the Smad2 protein. Smad2, in combination with Smad4, is translocated to the nucleus where the activated Smad complex recruits other transcription factors (TF) that together activate the expression of target genes that mediate the biological effects of TGF-?. Some of the activated target genes stimulate tumorigenesis, while others suppress tumorigenesis. References Kawabata, M., and Miyazono, K., Signal transduction of the TGF-? superfamily by Smad proteins. J. Biochem. (Tokyo), 125, 9-16 (1999). Wrana, J.L., TGF-? receptors and signalling mechanisms. Miner. Electrolyte Metab., 24, 120-130 (1998). Markowitz, S.D., and Roberts, A.B., Tumor suppressor activity of the TGF-? pathway in human cancers. Cytokine Growth Factor Rev., 7, 93-102 (1996).

68. Signalwege �ber regulierte Proteolyse In vielen Entwicklungsprozessen werden Signalwege genutzt, die �ber regulierte Proteolyse verlaufen. Die meisten Signalwege wurden in Drosophila entdeckt, sind jedoch in der Evolution stark konserviert. Beispiele: Signalweg �ber Notch-Rezeptor Wnt Signalweg Hedgehog Signalweg NF-?B Signalweg

69. Wnt Signalweg Wnt Proteine werden sekretiert, kontrollieren Entwicklung. �-Catenin ist normalerweise �ber Cadherin an Zell-Verbindungen lokalisiert. Freies cytosolisches �-Catenin wird �ber Degradationkomplex abgebaut: Glykogensynthase Kinase-3� (GSK-3�) phosphoryliert �-Catenin. Tumorsuppressor-Protein adenomatous polyposis coli (APC) erh�ht Affinit�t. Axin: Ger�stprotein Bindung von Wnt an Rezeptor Frizzled und Co-Rezeptor LRP (LDL-receptor-related protein) aktiviert Dishevelled (Dsh). Dsh inhibiert GSK-3�. �-Catenin wandert in den Kern, aktiviert Gene, z.B. c-myc. In 80% aller Darmkrebsf�lle ist APC mutiert, so dass �-Catenin auch ohne Wnt Signal c-myc und andere Gene stimuliert. Presenilin-1 (PS1) is associated with gamma secretase activity that cleaves amyloid precursor protein (APP) and is implicated in Alzheimer's disease. Presenilin-1 is also a component in gamma-secretase activity involved in signaling by the transmembrane protein Notch. Active gamma secretase requires PS-1 N-terminal fragment and a C-terminal fragment and is unique in catalyzing proteolysis within the transmembrane region of proteins. Other proteins such as nicastrin may also be components of the gamma-secretase. Binding of the ligand Delta by Notch appears to trigger two proteolytic cleavages of Notch. The first step cleaves an extracellular domain and is catalyzed by a metalloprotease termed alpha-secretase or TACE. The second cleavage step appears to occur within the transmembrane domain of Notch, and releases a Notch intracellular doman (NICD). Once released, NICD moves into the nucleus where it is involved in transcriptional regulation through CSL family transcription factors (CBF1, Su(H), Lag-1) or other transcriptional regulators such as LEF-1. Presenilin is also involved in the Wnt/frizzled signaling pathway (see WNT signaling pathway) through beta-catenin. Beta-catenin is a cytoskeletal component that enters the nucleus to act as a transcriptional cofactor. Binding of WNT to Frizzled causes disheveled (DSH) to inhibit Glycogen synthase kinase 3 beta (GSK-3b) activity. Phosphorylation of Beta-catenin induces the ubiquitination and proteolytic degradation of beta-catenin by the proteasome. Non-phosphorylated beta-catenin is stable and enters the nucleus to regulate transcription with TCF. The beta-catenin/TCF complex activates genes that promote cellular survival, proliferation and differentiation during development. Presenilin stimulates beta-catenin turnover, reducing its transcriptional activation. Presenilin-1 (PS1) is associated with gamma secretase activity that cleaves amyloid precursor protein (APP) and is implicated in Alzheimer's disease. Presenilin-1 is also a component in gamma-secretase activity involved in signaling by the transmembrane protein Notch. Active gamma secretase requires PS-1 N-terminal fragment and a C-terminal fragment and is unique in catalyzing proteolysis within the transmembrane region of proteins. Other proteins such as nicastrin may also be components of the gamma-secretase. Binding of the ligand Delta by Notch appears to trigger two proteolytic cleavages of Notch. The first step cleaves an extracellular domain and is catalyzed by a metalloprotease termed alpha-secretase or TACE. The second cleavage step appears to occur within the transmembrane domain of Notch, and releases a Notch intracellular doman (NICD). Once released, NICD moves into the nucleus where it is involved in transcriptional regulation through CSL family transcription factors (CBF1, Su(H), Lag-1) or other transcriptional regulators such as LEF-1.

70. TNF/NF-?B Signalweg Bindung von TNF (tumor necrosis factor) an TNF Rezeptor vermittelt wachstumsregulatorische Signale in die Zelle. In normalen Zellen ist TNF mitogen, in Krebszellen initiiert TNF Apoptose. Aktivierung von NF-?B (p50/p65) durch Phosphorylierung (Ubiquitinylierung, Abbau) des Inhibitors I?B. Dissoziation von I?B setzt Kernsignalsequenz in NF-?B frei. NF-?B wandert in den Kern und stimuliert Transkritpion spezifischer Gene. In Zellen mit blockiertem NF-?B Signalweg wird durch TNF h�ufiger der apoptotische Signalweg angeregt. TNF als Anti-Tumor Wirkstoff? TNF Signaling Pathway When bound to tumor necrosis factor (TNF), the TNF receptor (TNFR) (55 kDa) transduces growth regulatory signals into the cell. TNF is mitogenic in normal cells; however, TNF initiates programmed cell death (PCD) or apoptosis in transformed cells causing DNA fragmentation and cytolysis. Functional studies have identified a conserved region within the receptor, termed the death domain (DD), a protein-protein interaction motif that is necessary to transmit the apoptotic signal. The TNF-induced survival pathway is mediated by the transcription factor NF-?B. Activation of NF- ?B occurs via phosphorylation of I ?B at Ser32 and Ser36, resulting in the dissociation and subsequent nuclear localization of active NF- ?B. Recent studies have demonstrated that cells in which the NF- ?B signaling pathway is blocked are more likely to undergo apoptosis in response to TNF. Therefore, the availability of NF- ?B may play a critical role in the ability of TNF to act as an apoptosis-inducer and anti-tumor agent. References Plumpe, J., et al., NF- ?B determines between apoptosis and proliferation in hepatocytes during liver regeneration. Am. J. Physiol. Gastrointest. Liver Physiol., 278, G173-G183 (2000). Pimentel-Muinos, F.X., and Seed, B., Regulated commitment of TNF receptor signaling: a molecular switch for death or activation. Immunity, 11, 783-793 (1999). Schwandner, R., et al., TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase. J. Biol. Chem., 273, 5916-5922 (1998). TNF Signaling Pathway When bound to tumor necrosis factor (TNF), the TNF receptor (TNFR) (55 kDa) transduces growth regulatory signals into the cell. TNF is mitogenic in normal cells; however, TNF initiates programmed cell death (PCD) or apoptosis in transformed cells causing DNA fragmentation and cytolysis. Functional studies have identified a conserved region within the receptor, termed the death domain (DD), a protein-protein interaction motif that is necessary to transmit the apoptotic signal. The TNF-induced survival pathway is mediated by the transcription factor NF-?B. Activation of NF- ?B occurs via phosphorylation of I ?B at Ser32 and Ser36, resulting in the dissociation and subsequent nuclear localization of active NF- ?B. Recent studies have demonstrated that cells in which the NF- ?B signaling pathway is blocked are more likely to undergo apoptosis in response to TNF. Therefore, the availability of NF- ?B may play a critical role in the ability of TNF to act as an apoptosis-inducer and anti-tumor agent. References Plumpe, J., et al., NF- ?B determines between apoptosis and proliferation in hepatocytes during liver regeneration. Am. J. Physiol. Gastrointest. Liver Physiol., 278, G173-G183 (2000). Pimentel-Muinos, F.X., and Seed, B., Regulated commitment of TNF receptor signaling: a molecular switch for death or activation. Immunity, 11, 783-793 (1999). Schwandner, R., et al., TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase. J. Biol. Chem., 273, 5916-5922 (1998).

71. Beispiel: Insulin Signalweg Insulinbindung bewirkt Autophosphorylierung des Insulin-Rezeptors und Phosphorylierung von IRS (insulin receptor substrates). IRS phsophoryliert SH3-Dom�ne von Adaptormolek�l Grb2. Aktiviertes Grb2 rekrutiert Sos und aktiviert Ras-Signalweg und Gentranskription. IRS aktiviert PI3-Kinase und erh�ht damit Konzentration von PIP2 und PIP, was PDK1 aktiviert. PDK1 aktiviert Akt/PKB, was zu einer Translokation des Glukose-Transporters (GLUT4) zur Zellmembran f�hrt. Insulin Pathway Signaling through the insulin pathway is critical for the regulation of intracellular and blood glucose levels and the avoidance of diabetes. Insulin binds to its receptor leading to the autophosphorylation of the �-subunits and the tyrosine phosphorylation of insulin receptor substrates (IRS). IRS phosphorylates the SH2 domain of Shp2, a tyrosine phosphatase, and the SH3 domain of the adaptor molecule Grb2. Activated Grb2 recruits Sos1 that, in turn, activates the Ras signaling pathway and gene transcription. IRS also activates phosphoinositide 3-kinase (PI3K) through its SH2 domain, thus increasing the intracellular concentration of PIP2 and PIP. This, in turn, activates phosphatidylinositol phosphate-dependent kinase-1 (PDK-1), that subsequently activates Akt/PKB This results in the translocation of the glucose transporter (GLUT4) from cytoplasmic vesicles to the cell membrane. References Bevan, P., Insulin signaling. J. Cell Sci., 114, 1429-1430 (2001). Kido, Y., et al., Clinical review 125: The insulin receptor and its cellular targets. J. Clin. Endocrinol. Metab., 86, 972-979 (2001). Insulin Pathway Signaling through the insulin pathway is critical for the regulation of intracellular and blood glucose levels and the avoidance of diabetes. Insulin binds to its receptor leading to the autophosphorylation of the �-subunits and the tyrosine phosphorylation of insulin receptor substrates (IRS). IRS phosphorylates the SH2 domain of Shp2, a tyrosine phosphatase, and the SH3 domain of the adaptor molecule Grb2. Activated Grb2 recruits Sos1 that, in turn, activates the Ras signaling pathway and gene transcription. IRS also activates phosphoinositide 3-kinase (PI3K) through its SH2 domain, thus increasing the intracellular concentration of PIP2 and PIP. This, in turn, activates phosphatidylinositol phosphate-dependent kinase-1 (PDK-1), that subsequently activates Akt/PKB This results in the translocation of the glucose transporter (GLUT4) from cytoplasmic vesicles to the cell membrane. References Bevan, P., Insulin signaling. J. Cell Sci., 114, 1429-1430 (2001). Kido, Y., et al., Clinical review 125: The insulin receptor and its cellular targets. J. Clin. Endocrinol. Metab., 86, 972-979 (2001).

72. Beispiel: EGF Rezeptor Signalweg Mitglieder der EGF-Rezeptorfamilie (EGFR, ErbB2, ErbB3, ErbB4) bilden verschiedene Homo- und Heterodimere, die verschiedene Liganden binden und verschiedene Signalwege stimulieren. EGF Receptor Signal Transduction Pathway The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four receptors, EGF-R (ErbB1), ErbB2 (Neu), ErbB3, and ErbB4. Members of the EGF-R family contain a cytoplasmic tyrosine kinase domain, a single transmembrane domain, and an extracellular domain that is involved in ligand binding and receptor dimerization. Activation of the EGF-R results in the initiation of a diverse array of cellular pathways. In response to toxic environmental stimuli, such as ultraviolet irradiation, or to receptor occupation by EGF, the EGF-R forms homo- or heterodimers with other family members. Each dimeric receptor complex will initiate a distinct signaling pathway by recruiting different Src homology 2 (SH2)-containing effector proteins. Dimerization results in autophosphorylation initiating a downstream cascade of events culminating in cellular responses such as cell proliferation or apoptosis. The activated EGF-R dimer complexes with the adaptor protein, Grb, coupled to the guanine nucleotide releasing factor, SOS. The Grb-SOS complex can either bind directly to phosphotyrosine sites in the receptor or indirectly through Shc. These protein interactions bring SOS in close proximity to Ras, allowing for Ras activation. This subsequently activates the ERK and JNK signaling pathways that, in turn, activate transcription factors, such as c-fos, AP-1, and Elk-1, that promote gene expression and contribute to cell proliferation. References Daly, R.J., Take your partners, please - signal diversification by the erbB family of receptor tyrosine kinases. Growth Factors, 16, 255-263 (1999). Wells, A., EGF receptor. Int. J. Biochem. Cell Biol., 31, 637-643 (1999). Rosette, C., Karin, M., Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science, 274, 1194-1197 1996). Qian, X., et al., N terminus of Sos1 Ras exchange factor: critical roles for the Dbl and pleckstrin homology domains. Mol. Cell Biol., 18, 771-778 (1998). EGF Receptor Signal Transduction Pathway The epidermal growth factor (EGF) family of receptor tyrosine kinases consists of four receptors, EGF-R (ErbB1), ErbB2 (Neu), ErbB3, and ErbB4. Members of the EGF-R family contain a cytoplasmic tyrosine kinase domain, a single transmembrane domain, and an extracellular domain that is involved in ligand binding and receptor dimerization. Activation of the EGF-R results in the initiation of a diverse array of cellular pathways. In response to toxic environmental stimuli, such as ultraviolet irradiation, or to receptor occupation by EGF, the EGF-R forms homo- or heterodimers with other family members. Each dimeric receptor complex will initiate a distinct signaling pathway by recruiting different Src homology 2 (SH2)-containing effector proteins. Dimerization results in autophosphorylation initiating a downstream cascade of events culminating in cellular responses such as cell proliferation or apoptosis. The activated EGF-R dimer complexes with the adaptor protein, Grb, coupled to the guanine nucleotide releasing factor, SOS. The Grb-SOS complex can either bind directly to phosphotyrosine sites in the receptor or indirectly through Shc. These protein interactions bring SOS in close proximity to Ras, allowing for Ras activation. This subsequently activates the ERK and JNK signaling pathways that, in turn, activate transcription factors, such as c-fos, AP-1, and Elk-1, that promote gene expression and contribute to cell proliferation. References Daly, R.J., Take your partners, please - signal diversification by the erbB family of receptor tyrosine kinases. Growth Factors, 16, 255-263 (1999). Wells, A., EGF receptor. Int. J. Biochem. Cell Biol., 31, 637-643 (1999). Rosette, C., Karin, M., Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors. Science, 274, 1194-1197 1996). Qian, X., et al., N terminus of Sos1 Ras exchange factor: critical roles for the Dbl and pleckstrin homology domains. Mol. Cell Biol., 18, 771-778 (1998).

73. Calcium Signalgebung

74. Ca2+ als intrazellul�rer Botenstoff Beispiele: Ca2+ Welle nach Befruchtung einer Eizelle startet embryonale Entwicklung. Ca2+ bewirkt Kontraktion von Muskelzellen. Ca2+ bewirkt Sekretion in vielen sekretorischen Zellen, inklusive Nervenzellen. Fertilization of an egg by a sperm triggering an increase in cytosolic Ca2+. This starfish egg was injected with a Ca2+-sensitive fluorescent dye before it was fertilized. A wave of cytosolic Ca2+(red), released from the endoplasmic reticulum, is seen to sweep across the egg from the site of sperm entry (arrow). This Ca2+ wave provokes a change in the egg cell surface, preventing the entry of other sperm, and it also initiates embryonic development (discussed in Chapter 20). (Courtesy of Stephen A. Stricker.) The main ways eucaryotic cells maintain a very low concentration of free Ca2+in their cytosol. (A) Ca2+ is actively pumped out of the cytosol to the cell exterior. (B) Ca2+ is pumped into the ER and mitochondria, and various molecules in the cell bind free Ca2+ tightly. Fertilization of an egg by a sperm triggering an increase in cytosolic Ca2+. This starfish egg was injected with a Ca2+-sensitive fluorescent dye before it was fertilized. A wave of cytosolic Ca2+(red), released from the endoplasmic reticulum, is seen to sweep across the egg from the site of sperm entry (arrow). This Ca2+ wave provokes a change in the egg cell surface, preventing the entry of other sperm, and it also initiates embryonic development (discussed in Chapter 20). (Courtesy of Stephen A. Stricker.) The main ways eucaryotic cells maintain a very low concentration of free Ca2+in their cytosol. (A) Ca2+ is actively pumped out of the cytosol to the cell exterior. (B) Ca2+ is pumped into the ER and mitochondria, and various molecules in the cell bind free Ca2+ tightly.

75. Ca2+ als intrazellul�rer Botenstoff Fertilization of an egg by a sperm triggering an increase in cytosolic Ca2+. This starfish egg was injected with a Ca2+-sensitive fluorescent dye before it was fertilized. A wave of cytosolic Ca2+(red), released from the endoplasmic reticulum, is seen to sweep across the egg from the site of sperm entry (arrow). This Ca2+ wave provokes a change in the egg cell surface, preventing the entry of other sperm, and it also initiates embryonic development (discussed in Chapter 20). (Courtesy of Stephen A. Stricker.) The main ways eucaryotic cells maintain a very low concentration of free Ca2+in their cytosol. (A) Ca2+ is actively pumped out of the cytosol to the cell exterior. (B) Ca2+ is pumped into the ER and mitochondria, and various molecules in the cell bind free Ca2+ tightly. Fertilization of an egg by a sperm triggering an increase in cytosolic Ca2+. This starfish egg was injected with a Ca2+-sensitive fluorescent dye before it was fertilized. A wave of cytosolic Ca2+(red), released from the endoplasmic reticulum, is seen to sweep across the egg from the site of sperm entry (arrow). This Ca2+ wave provokes a change in the egg cell surface, preventing the entry of other sperm, and it also initiates embryonic development (discussed in Chapter 20). (Courtesy of Stephen A. Stricker.) The main ways eucaryotic cells maintain a very low concentration of free Ca2+in their cytosol. (A) Ca2+ is actively pumped out of the cytosol to the cell exterior. (B) Ca2+ is pumped into the ER and mitochondria, and various molecules in the cell bind free Ca2+ tightly.

76. Calmodulin (CaM) bindet Ca2+ Wichtigstes Ca2+ bindendes Protein. Vier hochaffine Ca2+ Bindungsstellen (EF-Hand-Bindungsmotiv). Allosterische Konformations�nderung nach Bindung von = 2 Ca2+. EF Hand. Formed by a helix-loop-helix unit, an EF hand is a binding site for calcium in many calcium sensing proteins. Here, the E helix is yellow, the F helix is blue, and calcium is represented by the green sphere. EF Hand. Formed by a helix-loop-helix unit, an EF hand is a binding site for calcium in many calcium sensing proteins. Here, the E helix is yellow, the F helix is blue, and calcium is represented by the green sphere.

77. Ca2+/CaM-abh�ngige Kinasen (CaM Kinasen) CaM zeigt �Klappmesser�-�hnliche Konformations�nderung bei Bindung der Substrathelix. An amphipatic a helix (purple) in CaM kinase I is a target for calmodulin. (B) After calcium binding (1), the two halves of calmodulin clamp down around the target helix (2), binding it through hydrophobic and ionic interactions. In CaM kinase I, this interaction extracts a C-terminal a helix, allowing the enzyme to adopt an active conformation. The structure of Ca2+/calmodulin based on x-ray diffraction and NMR studies. (A) The molecule has a "dumbbell" shape, with two globular ends connected by a long, exposed a helix. Each end has two Ca2+-binding domains, each with a loop of 12 amino acids, in which aspartic acid and glutamic acid side chains form ionic bonds with Ca2+. The two Ca2+-binding sites in the carboxyl-terminal part of the molecule have a tenfold higher affinity for Ca2+ than the two in the amino-terminal part. In solution, the molecule is flexible, displaying a range of forms, from extended (as shown) to more compact. (B) The major structural change in Ca2+/calmodulin that occurs when it binds to a target protein (in this example, a peptide that consists of the Ca2+/calmodulin-binding domain of a Ca2+/calmodulin-dependent protein kinase). Note that the Ca2+/calmodulin has "jack-knifed" to surround the peptide. (A, based on x-ray crystallographic data from Y.S. Babu et al., Nature 315:37 40, 1985; B, based on x-ray crystallographic data from W.E. Meador, A.R. Means, and F.A. Quiocho, Science 257:1251 1255, 1992, and on NMR data from M. Ikura et al., Science 256:632 638, 1992. � AAAS.) An amphipatic a helix (purple) in CaM kinase I is a target for calmodulin. (B) After calcium binding (1), the two halves of calmodulin clamp down around the target helix (2), binding it through hydrophobic and ionic interactions. In CaM kinase I, this interaction extracts a C-terminal a helix, allowing the enzyme to adopt an active conformation. The structure of Ca2+/calmodulin based on x-ray diffraction and NMR studies. (A) The molecule has a "dumbbell" shape, with two globular ends connected by a long, exposed a helix. Each end has two Ca2+-binding domains, each with a loop of 12 amino acids, in which aspartic acid and glutamic acid side chains form ionic bonds with Ca2+. The two Ca2+-binding sites in the carboxyl-terminal part of the molecule have a tenfold higher affinity for Ca2+ than the two in the amino-terminal part. In solution, the molecule is flexible, displaying a range of forms, from extended (as shown) to more compact. (B) The major structural change in Ca2+/calmodulin that occurs when it binds to a target protein (in this example, a peptide that consists of the Ca2+/calmodulin-binding domain of a Ca2+/calmodulin-dependent protein kinase). Note that the Ca2+/calmodulin has "jack-knifed" to surround the peptide. (A, based on x-ray crystallographic data from Y.S. Babu et al., Nature 315:37 40, 1985; B, based on x-ray crystallographic data from W.E. Meador, A.R. Means, and F.A. Quiocho, Science 257:1251 1255, 1992, and on NMR data from M. Ikura et al., Science 256:632 638, 1992. � AAAS.)

78. Bindung von pTyr in aktivierten Rezeptor-Tyrosinkinasen (RTK) Viele verschiedene Proteine binden �ber SH2- oder PTB-Dom�ne an pTyr aktivierter RTKs.



81. Exkurs: EF-Hand-Dom�ne bindet Ca2+ EF Hand. Formed by a helix-loop-helix unit, an EF hand is a binding site for calcium in many calcium sensing proteins. Here, the E helix is yellow, the F helix is blue, and calcium is represented by the green sphere. The EF-Hand DomainStructure The basic EF-hand consists of two perpendicular 10 to 12 residue alpha helices with a 12-residue loop region between, forming a single calcium-binding site (helix-loop-helix). Calcium ions interact with residues contained within the loop region. Each of the 12 residues in the loop region is important for calcium coordination. In EF-hand motifs, residues 1, 3, 5, 7, 9, and 12 of the conserved loop region provide oxygen ligands to the calcium ion necessary for its binding. In most EF-hand proteins the residue at position 12 is a glutamate. The glutamate contributes both its side-chan oxygens for calcium coordination. Variations in calcium binding affinity for different EF-hand proteins is due to the amino-acid composition in the 1, 3, 5, 7, and 9 positions. Domain Binding and FunctionThe EF-hand motif contains approximately 40 residues and is involved in binding intracellular calcium. EF-hand domains are often found in single or multiple pairs, giving rise to various structural/functional variations in proteins containing EF-hand motifs. Proteins containing EF-hands can be grouped into two functional categories ? regulatory or structural. Binding of calcium to regulatory EF hand domain-containing proteins induces a conformational change, which is transmitted to their target proteins, often catalyzing enzymatic reactions. In contrast, binding of calcium to structural EF-hand domain-containing proteins does not induce a significant conformational change. Structural EF-hand domains seem to play a role in buffering intracellular calcium levels. Binding ExamplesEF-hand proteins Binding partner Function Calmodulin Ca2+ Regulatory proteins S-100 Ca2+ Regulatory proteins recoverin Ca2+ Regulatory proteins calbindin Ca2+ Structural proteins parvalbumin Ca2+ Structural proteins EF Hand. Formed by a helix-loop-helix unit, an EF hand is a binding site for calcium in many calcium sensing proteins. Here, the E helix is yellow, the F helix is blue, and calcium is represented by the green sphere. The EF-Hand DomainStructure The basic EF-hand consists of two perpendicular 10 to 12 residue alpha helices with a 12-residue loop region between, forming a single calcium-binding site (helix-loop-helix). Calcium ions interact with residues contained within the loop region. Each of the 12 residues in the loop region is important for calcium coordination. In EF-hand motifs, residues 1, 3, 5, 7, 9, and 12 of the conserved loop region provide oxygen ligands to the calcium ion necessary for its binding. In most EF-hand proteins the residue at position 12 is a glutamate. The glutamate contributes both its side-chan oxygens for calcium coordination. Variations in calcium binding affinity for different EF-hand proteins is due to the amino-acid composition in the 1, 3, 5, 7, and 9 positions. Domain Binding and FunctionThe EF-hand motif contains approximately 40 residues and is involved in binding intracellular calcium. EF-hand domains are often found in single or multiple pairs, giving rise to various structural/functional variations in proteins containing EF-hand motifs. Proteins containing EF-hands can be grouped into two functional categories ? regulatory or structural. Binding of calcium to regulatory EF hand domain-containing proteins induces a conformational change, which is transmitted to their target proteins, often catalyzing enzymatic reactions. In contrast, binding of calcium to structural EF-hand domain-containing proteins does not induce a significant conformational change. Structural EF-hand domains seem to play a role in buffering intracellular calcium levels. Binding ExamplesEF-hand proteins Binding partner Function Calmodulin Ca2+ Regulatory proteins S-100 Ca2+ Regulatory proteins recoverin Ca2+ Regulatory proteins calbindin Ca2+ Structural proteins parvalbumin Ca2+ Structural proteins

82. Exkurs: PH-Dom�ne bindet Inositolphosphat The PH DomainStructure Despite unusually divergent primary sequences, PH domains share a conserved fold made up of a b-barrel composed of two roughly perpendicular, anti-parallel beta-sheets and a C-terminal alpha amphipathic -helix. PH domains bind to their inositol phosphate ligands via a binding surface composed primarily of residues from the beta1/beta2, beta3/beta4, and beta6/beta7 loops. Basic residues in the beta1-beta2 loop are especially important for binding the phosphatidylinositide phosphates, by establsihing a positive electric potential on the face of the PH domain. Comparison of different PH domains with distinct phosphoinosite binding specificities has revealed insights into what structural features dictate binding specificity. For example, a tyrosine residue located in the beta-strand is strictly conserved in PH domains that bind PI3- kinase products with high affinity but not in other PH domains. Interestingly, PH domains have a strikingly similar structure to PTB domains, the Enabled / Vasp homology (EVH1/WH1) and the Ran-binding domain (RanBD) of RBP2, despite the absence of any sequence homology. The figure shows the complex between the PH domain of Phospholipase C-d and inositol-(1,4,5)-trisphosphate (red). Domain Binding and FunctionPleckstrin-homology (PH) domains are protein modules of approximately 120 amino acids found in a wide variety of signalling proteins in organisms ranging for yeast to humans. Some PH domains bind with high affinity (low mM or nM Kd) to specific phosphoinositides such as phosphatidyl-inositol (PI) -4,5-bisphosphate, PI-3,4-P2 or PI-3,4,5-P3. Binding to phosphoinositides may allow PH proteins to respond to lipid messengers for example by relocation to membranes. The C-termini of some PH domains have also been reported to bind the beta/lambda subunits of heterotrimeric G proteins. Examples of Domain ProteinsPH domain Protein Specific Phosphoinositide Ligand Phospholipase C ; mSos1 PI-4,5-P2 Btk Tyr Kinase; Grp1 PI-3,4,5-P3 Akt/PKB Ser/Thr Kinase PI-3,4-P2 The PH DomainStructure Despite unusually divergent primary sequences, PH domains share a conserved fold made up of a b-barrel composed of two roughly perpendicular, anti-parallel beta-sheets and a C-terminal alpha amphipathic -helix. PH domains bind to their inositol phosphate ligands via a binding surface composed primarily of residues from the beta1/beta2, beta3/beta4, and beta6/beta7 loops. Basic residues in the beta1-beta2 loop are especially important for binding the phosphatidylinositide phosphates, by establsihing a positive electric potential on the face of the PH domain. Comparison of different PH domains with distinct phosphoinosite binding specificities has revealed insights into what structural features dictate binding specificity. For example, a tyrosine residue located in the beta-strand is strictly conserved in PH domains that bind PI3- kinase products with high affinity but not in other PH domains. Interestingly, PH domains have a strikingly similar structure to PTB domains, the Enabled / Vasp homology (EVH1/WH1) and the Ran-binding domain (RanBD) of RBP2, despite the absence of any sequence homology. The figure shows the complex between the PH domain of Phospholipase C-d and inositol-(1,4,5)-trisphosphate (red). Domain Binding and FunctionPleckstrin-homology (PH) domains are protein modules of approximately 120 amino acids found in a wide variety of signalling proteins in organisms ranging for yeast to humans. Some PH domains bind with high affinity (low mM or nM Kd) to specific phosphoinositides such as phosphatidyl-inositol (PI) -4,5-bisphosphate, PI-3,4-P2 or PI-3,4,5-P3. Binding to phosphoinositides may allow PH proteins to respond to lipid messengers for example by relocation to membranes. The C-termini of some PH domains have also been reported to bind the beta/lambda subunits of heterotrimeric G proteins. Examples of Domain ProteinsPH domain Protein Specific Phosphoinositide Ligand Phospholipase C ; mSos1 PI-4,5-P2 Btk Tyr Kinase; Grp1 PI-3,4,5-P3 Akt/PKB Ser/Thr Kinase PI-3,4-P2

83. Exkurs: C2-Dom�ne bindet Ca2+ und Phospholipide C2-Dom�ne: Zwei vierstr�ngige �-Faltbl�tter mit drei Schleifen oben und und vier Schleifen unten. F�nf konservierte Asp und ein Ser in den oberen Schleifen binden 3 Ca2+. Ca2+-abh�ngige oder �unabh�ngige Bindung von Phospholipiden. The C2 DomainStructure The C2 domain is composed of two, four stranded b-sheets creating three loops at the top of the domain and four at the bottom. Five conserved aspartate residues and one serine in upper loops 1 and 3 are involved in the binding of 3 calcium ions necessary for phospholipid binding. Analysis of the C2 domain from a number of different proteins reveals the C2 domains can exist in two topologies. In topology I (ie. C2A of synaptotagmin) the first b-strand occupies the same position as b-strand eight in topology II (ie. PLCd-1). The N and C terminus in topology I is oriented in the up position while in topology II the N and C terminus are oriented down. The functional significance of the two topologies remains unknown. Domain Binding and FunctionThe C2 domain is a region containing approximately 130 residues involved in binding phospholipids in a calcium dependent manner or calcium independent manner. C2 domains are found in over 100 different proteins with functions ranging from signal transduction to vesicular trafficking. Calcium binding to the C2 domain of synaptotagmin induces little conformational change in the C2 domain but rather calcium induces a change in the electrostatic potential enhancing phospholipid binding, suggesting the C2 domain functions as an electrostatic switch. In addition to electrostatic interactions, side chains in the calcium binding loops influence the binding of different C2 domains to either neutral or negatively charged phospholipids. Binding ExamplesC2 domain proteins Binding partner PKC b Ser/Thr Kinase Ca2+ and acidic phospholipids Synaptotagmin Integral membrane protein in synaptic vesicles Ca2+ and acidic phospholipids The C2 DomainStructure The C2 domain is composed of two, four stranded b-sheets creating three loops at the top of the domain and four at the bottom. Five conserved aspartate residues and one serine in upper loops 1 and 3 are involved in the binding of 3 calcium ions necessary for phospholipid binding. Analysis of the C2 domain from a number of different proteins reveals the C2 domains can exist in two topologies. In topology I (ie. C2A of synaptotagmin) the first b-strand occupies the same position as b-strand eight in topology II (ie. PLCd-1). The N and C terminus in topology I is oriented in the up position while in topology II the N and C terminus are oriented down. The functional significance of the two topologies remains unknown. Domain Binding and FunctionThe C2 domain is a region containing approximately 130 residues involved in binding phospholipids in a calcium dependent manner or calcium independent manner. C2 domains are found in over 100 different proteins with functions ranging from signal transduction to vesicular trafficking. Calcium binding to the C2 domain of synaptotagmin induces little conformational change in the C2 domain but rather calcium induces a change in the electrostatic potential enhancing phospholipid binding, suggesting the C2 domain functions as an electrostatic switch. In addition to electrostatic interactions, side chains in the calcium binding loops influence the binding of different C2 domains to either neutral or negatively charged phospholipids. Binding ExamplesC2 domain proteins Binding partner PKC b Ser/Thr Kinase Ca2+ and acidic phospholipids Synaptotagmin Integral membrane protein in synaptic vesicles Ca2+ and acidic phospholipids

84. Exkurs: SH2-Dom�ne bindet Phosphotyrosin The three-dimensional structure of an SH2 domain, as determined by x-ray crystallography. The binding pocket for phosphotyrosine is shown in yellow on the right, and a pocket for binding a specific amino acid side chain (isoleucine, in this case) is shown in yellow on the left (see also Figure 3-40). (C) The SH2 domain is a compact, "plug-in" module, which can be inserted almost anywhere in a protein without disturbing the protein's folding or function (see Figure 3-19). Because each domain has distinct sites for recognizing phosphotyrosine and for recognizing a particular amino acid side chain, different SH2 domains recognize phosphotyrosine in the context of different flanking amino acid sequences. (B, based on data from G. Waksman et al., Cell 72:1 20, 1993. � Elsevier.) The SH2 DomainStructure SH2 domains contain a central anti-parallel b-sheet surrounded by two a-helices. The phosphopeptide generally binds as an extended b-strand that lies at right angles to the SH2 b-sheet. Conserved residues contribute to the hydrophobic core or are involved in pY recognition while more variable residues contribute to specific recognition of C-terminal residues. An invariant Arg residue in the SH2 domain coordinates the phosphate oxygens of pY and is essential for high affinity phosphopeptide binding. The figure shows the SH2 domain of v-src bound to a pYEEI peptide ligand. Domain Binding and FunctionSrc-homology 2 (SH2) domains are modules of ~100 amino acids that bind to specific phospho (pY)-containing peptide motifs. Conventional SH2 domains have a conserved pocket that recognizes pY, and a more variable pocket that binds 3-6 residues C-terminal to the pY and confers specificity. The SAP SH2 domain recognizes Y as well as pY in the context of residues N and C terminal, suggesting an alternate 3-pronged model may apply in some cases. Phosphopeptides of optimal sequence bind to SH2 domains with dissociation constants of ~50-500 nM. Examples of domain proteinsThe modular SH2 domain is found embedded in a wide variety of metazoan proteins that regulate functionally diverse processes. The figure below indicates the domain organization of representative members from various SH2 domain containing protein families. (Reference: Pawson, T., Gish, G., and Nash, P. Trends in Cell Biology Vol.11 No.12 December 2001) Binding properties of SH2 domainsConventional SH2 domains must achieve something of a balancing act. Their affinity for an unphosphorylated site must not be too high, or binding will not be regulated by phosphorylation. At the same time, the SH2 domain must obtain sufficient binding energy from the recognition of adjacent residues to allow discrimination between different phosphorylated sites, and thus a degree of specificity. Furthermore, SH2 domains must exhibit sufficiently high off-rates for rapid and reversible signal transduction. A structural basis for the specificity of SH2 domain-mediated interactions has been provided by numerous crystal and solution structures of SH2 domains bound to specific phosphotyrosine-containing peptides (sKuriyan J, Cowburn D. Annu Rev Biophys Biomol Struct 1997. 26, 259-288). The SH2 domain fold, which is composed of a central anti-parallel b-sheet sandwiched between two a-helices, provides a positively charged pocket on one side of the b-sheet for binding of the ligands phosphotyrosine moiety, and an extended surface on the other for binding to ligand residues C-terminal to the phosphotyrosine. Ribbons diagram of the SH2-C domain of phospholipase-C( bound to a specific phosphotyrosine-containing peptide. The peptide (gold) is derived from the cytoplasmic domain of the PDGF receptor (DNDpYIPLPDPK) derived from PTB:2PLD (Pascal SM, Singer AU, Gish G et al. Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma1 complexed with a high affinity binding peptide. Cell 1994. 77, 461-472.). Subtle differences in the molecular architecture of this extended surface can have very significant effects on ligand-binding specificity. For example, mutation of a threonine residue in this region of the Src SH2 domain to tryptophan, found at the corresponding position of the Grb2 SH2 domain, converted ligand-binding specificity from the Src-like pTyr-Glu-Glu-Ile, to the signature Grb2 binding motif pTyr-X-Asn (Kimber MS, Nachman J, Gish G, Pawson T, Pai E. Molecular Cell 2000. 5, 1043-1049). This mutant Src SH2 domain behaves biologically like a Grb2 SH2 domain, and not like a Src SH2 domain, indicating that biological activity tracks with biochemical binding specificity. Structural analysis provides a basis for understanding SH2 domain binding specificity. Crystal structures of the Grb2 and Src SH2 domains bound to optimal phosphopeptides illustrate the different modes of ligand binding. A switch in the ligand preference of the Src SH2 domain to pTyr-Val-Asn-Val is achieved through mutation of a threonine residue, highlighted in blue on the surface of the Src SH2, to the tryptophan shown as red in the Src T215W SH2 domain. In a similar fashion, the molecular architecture of the Grb2 SH2 domain uses a tryptophan (red) to define its binding to pTyr-X-Asn ligands. (For more details see Pawson, T., Gish, G., and Nash, P. Trends in Cell Biology Vol.11 No.12 December 2001) The PTB DomainStructure The PTB domains of Shc and IRS-1 contain two orthogonal b-sheets and connecting loops, and have very similar folds despite their low sequence similarity. Both have a C-terminal amphipathic a-helix capping one end of the b-sandwich. The N-terminal residues of the peptide ligand form an additional anti-parallel b-strand to the second b-sheet. The figure shows the PTB domain of Shc complexed to a HIIENPQpYFS peptide Domain Binding and FunctionPhosphotyrosine binding (PTB) domains are 100-150 residue modules that commonly bind Asn-Pro-X-Tyr motifs. The PTB domains of the docking proteins Shc and IRS-1 require ligand phosphorylation on the tyrosine residue (NPXpY) for binding. More N-terminal sequences are also required for high affinity binding and conferring specificity. The peptide binds as a b-strand to an anti-parallel b-sheet, while the NPXpY motif makes a turn, positioning the pY for recognition by basic residues. The PTB domains of proteins such as X11, Dab, Fe65 and Numb apparently recognize NPXY or related peptide motifs, but are not dependent on ligand phosphorylation. In addition, the Numb PTB domain can bind an unrelated peptide that forms a helical turn. Examples of Domain ProteinsPTB Domain Protein Binding Partner and Peptide Ligase Shc docking protein TrkA Nerve Growth Factor Receptor: Ile-Ile-Asn-Pro-Gln-pTyr IRS-1 docking protein Insulin receptor: Leu-Tyr-Ala-Ser-Ser-Asn-Pro-Glu-pTyr X11 neuronal protein b-amyloid precursor protein: Tyr-Glu-Asn-Pro-Thr-Tyr The three-dimensional structure of an SH2 domain, as determined by x-ray crystallography. The binding pocket for phosphotyrosine is shown in yellow on the right, and a pocket for binding a specific amino acid side chain (isoleucine, in this case) is shown in yellow on the left (see also Figure 3-40). (C) The SH2 domain is a compact, "plug-in" module, which can be inserted almost anywhere in a protein without disturbing the protein's folding or function (see Figure 3-19). Because each domain has distinct sites for recognizing phosphotyrosine and for recognizing a particular amino acid side chain, different SH2 domains recognize phosphotyrosine in the context of different flanking amino acid sequences. (B, based on data from G. Waksman et al., Cell 72:1 20, 1993. � Elsevier.) The SH2 DomainStructure SH2 domains contain a central anti-parallel b-sheet surrounded by two a-helices. The phosphopeptide generally binds as an extended b-strand that lies at right angles to the SH2 b-sheet. Conserved residues contribute to the hydrophobic core or are involved in pY recognition while more variable residues contribute to specific recognition of C-terminal residues. An invariant Arg residue in the SH2 domain coordinates the phosphate oxygens of pY and is essential for high affinity phosphopeptide binding. The figure shows the SH2 domain of v-src bound to a pYEEI peptide ligand. Domain Binding and FunctionSrc-homology 2 (SH2) domains are modules of ~100 amino acids that bind to specific phospho (pY)-containing peptide motifs. Conventional SH2 domains have a conserved pocket that recognizes pY, and a more variable pocket that binds 3-6 residues C-terminal to the pY and confers specificity. The SAP SH2 domain recognizes Y as well as pY in the context of residues N and C terminal, suggesting an alternate 3-pronged model may apply in some cases. Phosphopeptides of optimal sequence bind to SH2 domains with dissociation constants of ~50-500 nM. Examples of domain proteinsThe modular SH2 domain is found embedded in a wide variety of metazoan proteins that regulate functionally diverse processes. The figure below indicates the domain organization of representative members from various SH2 domain containing protein families. (Reference: Pawson, T., Gish, G., and Nash, P. Trends in Cell Biology Vol.11 No.12 December 2001) Binding properties of SH2 domainsConventional SH2 domains must achieve something of a balancing act. Their affinity for an unphosphorylated site must not be too high, or binding will not be regulated by phosphorylation. At the same time, the SH2 domain must obtain sufficient binding energy from the recognition of adjacent residues to allow discrimination between different phosphorylated sites, and thus a degree of specificity. Furthermore, SH2 domains must exhibit sufficiently high off-rates for rapid and reversible signal transduction. A structural basis for the specificity of SH2 domain-mediated interactions has been provided by numerous crystal and solution structures of SH2 domains bound to specific phosphotyrosine-containing peptides (sKuriyan J, Cowburn D. Annu Rev Biophys Biomol Struct 1997. 26, 259-288). The SH2 domain fold, which is composed of a central anti-parallel b-sheet sandwiched between two a-helices, provides a positively charged pocket on one side of the b-sheet for binding of the ligands phosphotyrosine moiety, and an extended surface on the other for binding to ligand residues C-terminal to the phosphotyrosine. Ribbons diagram of the SH2-C domain of phospholipase-C( bound to a specific phosphotyrosine-containing peptide. The peptide (gold) is derived from the cytoplasmic domain of the PDGF receptor (DNDpYIPLPDPK) derived from PTB:2PLD (Pascal SM, Singer AU, Gish G et al. Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma1 complexed with a high affinity binding peptide. Cell 1994. 77, 461-472.). Subtle differences in the molecular architecture of this extended surface can have very significant effects on ligand-binding specificity. For example, mutation of a threonine residue in this region of the Src SH2 domain to tryptophan, found at the corresponding position of the Grb2 SH2 domain, converted ligand-binding specificity from the Src-like pTyr-Glu-Glu-Ile, to the signature Grb2 binding motif pTyr-X-Asn (Kimber MS, Nachman J, Gish G, Pawson T, Pai E. Molecular Cell 2000. 5, 1043-1049). This mutant Src SH2 domain behaves biologically like a Grb2 SH2 domain, and not like a Src SH2 domain, indicating that biological activity tracks with biochemical binding specificity. Structural analysis provides a basis for understanding SH2 domain binding specificity. Crystal structures of the Grb2 and Src SH2 domains bound to optimal phosphopeptides illustrate the different modes of ligand binding. A switch in the ligand preference of the Src SH2 domain to pTyr-Val-Asn-Val is achieved through mutation of a threonine residue, highlighted in blue on the surface of the Src SH2, to the tryptophan shown as red in the Src T215W SH2 domain. In a similar fashion, the molecular architecture of the Grb2 SH2 domain uses a tryptophan (red) to define its binding to pTyr-X-Asn ligands. (For more details see Pawson, T., Gish, G., and Nash, P. Trends in Cell Biology Vol.11 No.12 December 2001) The PTB DomainStructure The PTB domains of Shc and IRS-1 contain two orthogonal b-sheets and connecting loops, and have very similar folds despite their low sequence similarity. Both have a C-terminal amphipathic a-helix capping one end of the b-sandwich. The N-terminal residues of the peptide ligand form an additional anti-parallel b-strand to the second b-sheet. The figure shows the PTB domain of Shc complexed to a HIIENPQpYFS peptide Domain Binding and FunctionPhosphotyrosine binding (PTB) domains are 100-150 residue modules that commonly bind Asn-Pro-X-Tyr motifs. The PTB domains of the docking proteins Shc and IRS-1 require ligand phosphorylation on the tyrosine residue (NPXpY) for binding. More N-terminal sequences are also required for high affinity binding and conferring specificity. The peptide binds as a b-strand to an anti-parallel b-sheet, while the NPXpY motif makes a turn, positioning the pY for recognition by basic residues. The PTB domains of proteins such as X11, Dab, Fe65 and Numb apparently recognize NPXY or related peptide motifs, but are not dependent on ligand phosphorylation. In addition, the Numb PTB domain can bind an unrelated peptide that forms a helical turn. Examples of Domain ProteinsPTB Domain Protein Binding Partner and Peptide Ligase Shc docking protein TrkA Nerve Growth Factor Receptor: Ile-Ile-Asn-Pro-Gln-pTyr IRS-1 docking protein Insulin receptor: Leu-Tyr-Ala-Ser-Ser-Asn-Pro-Glu-pTyr X11 neuronal protein b-amyloid precursor protein: Tyr-Glu-Asn-Pro-Thr-Tyr

85. Exkurs: PTB-Dom�ne bindet Phosphotyrosin The PTB DomainStructure The PTB domains of Shc and IRS-1 contain two orthogonal b-sheets and connecting loops, and have very similar folds despite their low sequence similarity. Both have a C-terminal amphipathic a-helix capping one end of the b-sandwich. The N-terminal residues of the peptide ligand form an additional anti-parallel b-strand to the second b-sheet. The figure shows the PTB domain of Shc complexed to a HIIENPQpYFS peptide Domain Binding and FunctionPhosphotyrosine binding (PTB) domains are 100-150 residue modules that commonly bind Asn-Pro-X-Tyr motifs. The PTB domains of the docking proteins Shc and IRS-1 require ligand phosphorylation on the tyrosine residue (NPXpY) for binding. More N-terminal sequences are also required for high affinity binding and conferring specificity. The peptide binds as a b-strand to an anti-parallel b-sheet, while the NPXpY motif makes a turn, positioning the pY for recognition by basic residues. The PTB domains of proteins such as X11, Dab, Fe65 and Numb apparently recognize NPXY or related peptide motifs, but are not dependent on ligand phosphorylation. In addition, the Numb PTB domain can bind an unrelated peptide that forms a helical turn. Examples of Domain ProteinsPTB Domain Protein Binding Partner and Peptide Ligase Shc docking protein TrkA Nerve Growth Factor Receptor: Ile-Ile-Asn-Pro-Gln-pTyr IRS-1 docking protein Insulin receptor: Leu-Tyr-Ala-Ser-Ser-Asn-Pro-Glu-pTyr X11 neuronal protein b-amyloid precursor protein: Tyr-Glu-Asn-Pro-Thr-Tyr The PTB DomainStructure The PTB domains of Shc and IRS-1 contain two orthogonal b-sheets and connecting loops, and have very similar folds despite their low sequence similarity. Both have a C-terminal amphipathic a-helix capping one end of the b-sandwich. The N-terminal residues of the peptide ligand form an additional anti-parallel b-strand to the second b-sheet. The figure shows the PTB domain of Shc complexed to a HIIENPQpYFS peptide Domain Binding and FunctionPhosphotyrosine binding (PTB) domains are 100-150 residue modules that commonly bind Asn-Pro-X-Tyr motifs. The PTB domains of the docking proteins Shc and IRS-1 require ligand phosphorylation on the tyrosine residue (NPXpY) for binding. More N-terminal sequences are also required for high affinity binding and conferring specificity. The peptide binds as a b-strand to an anti-parallel b-sheet, while the NPXpY motif makes a turn, positioning the pY for recognition by basic residues. The PTB domains of proteins such as X11, Dab, Fe65 and Numb apparently recognize NPXY or related peptide motifs, but are not dependent on ligand phosphorylation. In addition, the Numb PTB domain can bind an unrelated peptide that forms a helical turn. Examples of Domain ProteinsPTB Domain Protein Binding Partner and Peptide Ligase Shc docking protein TrkA Nerve Growth Factor Receptor: Ile-Ile-Asn-Pro-Gln-pTyr IRS-1 docking protein Insulin receptor: Leu-Tyr-Ala-Ser-Ser-Asn-Pro-Glu-pTyr X11 neuronal protein b-amyloid precursor protein: Tyr-Glu-Asn-Pro-Thr-Tyr

86. Exkurs: SH3-Dom�ne bindet prolinreiche Sequenzen The SH3 DomainStructure The basic fold of SH3 domains contains five anti-parallel beta-strands packed to form two perpendicular beta-sheets. The ligand-binding site consists of a hydrophobic patch that contains a cluster of conserved aromatic residues and is surrounded by two charged and variable loops. The figure shows a Sem5 C-terminal SH3 domain complexed to the mSos-derived sequence PPPVPPRRR. Domain Binding and FunctionSrc-homology 3 (SH3) domains generally bind to Pro-rich peptides that form a left-handed polyPro type II helix, with the minimal consensus Pro-X-X-Pro. Each Pro is usually preceded by an aliphatic residue. Each of these aliphatic-Pro pairs binds to a hydrophobic pocket on the SH3 domain. The detailed requirements of SH3 domain binding to its ligand have been examined by numerous approaches including phage display combinatorial peptide chemistry, nuclear magnetic resonance and crystal structure analysis. From this, two classes of SH3 domains have been defined (Class I and Class 2) which recognize RKXXPXXP and PXXPXR motifs respecitvely. The ligand can, in principle, bind in either orientation. Directionality is conferred by the interaction of the Arg or Lys residue with the charged outer face of the SH3 domain while the tandem prolines bind within two hydrophobic pockets of the SH3 domain. An additional non-Pro residue, frequently Arg, can form part of the binding core and contacts the SH3 domain. Such peptides usually bind to the SH3 domain with Kds in the mM range. The binding affinity and specificity can be markedly increased by tertiary interactions involving loops on the SH3 domain. In a few proteins, SH3 domains have been observed to bind in an unconventional non-PXXP manner. In these cases, either an alpha helical element or a tandem tyrosine motif interacts with a site on the SH3 domain that is either distinct or overlapping with the classical PXXP binding cleft. Examples of Domain ProteinsSH3 domain protein Binding partner SH3 domain binding site Src tyrosine kinase p85 subunit of PI 3-kinase RPLPVAPClass I N-terminal to C-terminal binding sit Crk adaptor protein C3G guanidine nucleotide exchanger PPPALPPKKRClass II C-terminal to N-terminal binding site FYB (FYN binding protein) SKAP55 Adaptor protein RKGDYASYunconventional Pex13p (integral peroxisomal membrane protein) Pex5p - PTS1 receptor WXXQFunconventional The SH3 DomainStructure The basic fold of SH3 domains contains five anti-parallel beta-strands packed to form two perpendicular beta-sheets. The ligand-binding site consists of a hydrophobic patch that contains a cluster of conserved aromatic residues and is surrounded by two charged and variable loops. The figure shows a Sem5 C-terminal SH3 domain complexed to the mSos-derived sequence PPPVPPRRR. Domain Binding and FunctionSrc-homology 3 (SH3) domains generally bind to Pro-rich peptides that form a left-handed polyPro type II helix, with the minimal consensus Pro-X-X-Pro. Each Pro is usually preceded by an aliphatic residue. Each of these aliphatic-Pro pairs binds to a hydrophobic pocket on the SH3 domain. The detailed requirements of SH3 domain binding to its ligand have been examined by numerous approaches including phage display combinatorial peptide chemistry, nuclear magnetic resonance and crystal structure analysis. From this, two classes of SH3 domains have been defined (Class I and Class 2) which recognize RKXXPXXP and PXXPXR motifs respecitvely. The ligand can, in principle, bind in either orientation. Directionality is conferred by the interaction of the Arg or Lys residue with the charged outer face of the SH3 domain while the tandem prolines bind within two hydrophobic pockets of the SH3 domain. An additional non-Pro residue, frequently Arg, can form part of the binding core and contacts the SH3 domain. Such peptides usually bind to the SH3 domain with Kds in the mM range. The binding affinity and specificity can be markedly increased by tertiary interactions involving loops on the SH3 domain. In a few proteins, SH3 domains have been observed to bind in an unconventional non-PXXP manner. In these cases, either an alpha helical element or a tandem tyrosine motif interacts with a site on the SH3 domain that is either distinct or overlapping with the classical PXXP binding cleft. Examples of Domain ProteinsSH3 domain protein Binding partner SH3 domain binding site Src tyrosine kinase p85 subunit of PI 3-kinase RPLPVAPClass I N-terminal to C-terminal binding sit Crk adaptor protein C3G guanidine nucleotide exchanger PPPALPPKKRClass II C-terminal to N-terminal binding site FYB (FYN binding protein) SKAP55 Adaptor protein RKGDYASYunconventional Pex13p (integral peroxisomal membrane protein) Pex5p - PTS1 receptor WXXQFunconventional

Signaltransduktion

Signaltransduktion

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Signaltransduktion WS 2008/2009

Zelluläre Signaltransduktion II

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