Signal Transduction

1. Signal Transduction 1 Reference: Molecular Biology of the cell 4th or 5th ed.

2. Why is signaling important? • Allows cells to respond to external stimuli such as: • Cytokines • Growth Factors • Hormones • Tissue Repair or Remodeling • Other cells • Stress • Tissue Specific Regulation • Regulate Differentiation and Development • Immune Response • Pathogens

3. Signaling is a process of cellular communication • Signals from outside to the inside result in changes to the cell: • Gene induction/suppression • Differentiation/Development • Protein secretion • Surface marker changes • Changes in cellular distribution • Environmental changes • Apoptosis • Proliferation • Motility • Destruction of foreign invaders • Destruction of aberrant cells

4. External signaling can be: Molecules involved in cell-cell and cell-matrix interactions. Autocrine signaling: signaling molecules released by a cell and only affect itself (e.g. many growth factors).

5. External signaling can be: • Paracrine signaling: signaling molecules released by a cell only affect target cells in close proximity to it (e.g. neurotransmitter and neurohormones). • Endocrine signaling: signaling molecules (hormones) synthesized by cells of the endocrine organs - act on target cells distant from their site of synthesis.

6. General model for signaling • Receptor – Ligand mediated signaling from outside to inside • Cascade of events (2nd Messengers --involving a number of different enzymes [Ca2+, kinases, phosphatases, adapter proteins, etc.) • Cellular Changes

8. Transmembrane Receptors Cell-surface receptors can be categorized into four classes: Receptor with intinsic enzymatic activity Tyrosine kinase receptors (EGF, insulin, PDGF) G-protein-coupled receptor e.g. receptors for epinephrine, serotonin and glucagon

9. Transmembrane Receptors Receptor with an associated enzyme e.g. cytokine receptors, receptors for growth hormone and interferons Receptor guanylylcyclases (atrialnatriuretic peptide) Ion-channel-linked receptor e.g. neurotransmitter-gated ion channels

10. TRANSDUCTION • Intracellular signaling pathways typically involve phosphorylation cascades that are reversibly and tightly controlled by protein kinases and protein phosphatases. • Kinases and phosphatases can be divided into: • transmembrane proteins or intracellular proteins. • serine/threonine-specific or tyrosine-specific (but also a class of dual-specific) • Tyrosine phosphorylation is rare in the cell • only <0.1% of total protein phosphorylation • But important in cellular regulation. • Their importance is evident from the fact that many protein tyrosine kinases (PTK) are encoded by proto-oncogenes

11. Receptor protein tyrosine kinase (RTKs)

12. Receptor protein tyrosine kinase (RTKs) • Extracellular region • Typically several hundred aa) • Ligand binds to the extracellular domain • Most known ligands are secreted soluble proteins • Membrane-bound and extracellular matrix-bound ligands can also activate receptor • Transmembrane region • All have a single hydrophobic transmembrane region followed by a few basic amino acids.)

13. Receptor protein tyrosine kinase (RTKs) • Cytoplasmic region • Contains a protein kinasecatalytic domain(PTK), • conserved in sequence of ~250 aa in length (conservation from 32-95%). • Contains a major tyrosine phosphorylation site (its phosphorylation is required for kinase activation in many cases) • A C-terminal region. • varies from a few up to 200 aa in length • most of the tyrosine phosphorylation occurs here. • Protein kinase activity is stimulated by binding of ligands to the extracellular side

14. P P P Receptor protein tyrosine kinase-initiated signal transduction • Ligand binding • Receptor oligomerization • tyrosine autophosphorylation of the receptor subunits

15. P P P P Signaling Kinase activation Binds other proteins Receptor protein tyrosine kinase-initiated signal transduction • Autophosphorylation of receptors serves two purposes: • activates catalytic activity of the PTK. • changes the conformation of the receptor that allows it to bind to next cytoplasmic signalling molecules in the cascade.

16. (I) Ligand binding induces receptor oligomerization • Ligand is a dimer • Ligands cluster on scaffolding • Ligands cluster in signaling cell • Ligands induce receptor-receptor interaction

17. A A A A B B B B B B B B a a a a a a a a b b b b • Examples: PDGF (platelet-derived growth factor) • PDGF are dimeric - homodimers or heterodimers of A and B chains. • PDGF A chain binds only a PDGF receptor • B chain binds both a and b receptors • Different composition of the PDGF appears to have different cellular responses.

18. Apart from the ligands, the extracellular domains of the receptors are also involved in the dimer formation • Examples: EGF (epidermal growth factor) • Ligands are monomeric. • Ligands induce both homo- and heterodimers of their receptors.

19. (II) Tyrosine phosphorylation of receptors • Ligand binding  Receptor oligomerization  Juxtapositioning of the cytoplasmic domains of the receptors  Conformational changes

20. The conformational changes allow Mg2+-ATP to bind the major autophosphorylation site in PTK (normally buried in the active site) • initial trans-phosphorylation occurs on a tyrosine residue in the other monomer of the receptor complex (Tyr857 in PDGF receptor) P P

21. Other Tyr residues in the receptors can now be phosphorylated by the activated receptor PTK • These phosphorylations serve as molecular switches to specifically bind cytoplasmic signaling molecules P P P P P P P P

22. Grb2 SH2 SH3 P P P P (III) Interaction of receptors with cytoplasmic proteins • The next step in PTK-mediated signaling involves interaction with cytoplasmic proteins that contain protein-protein interaction modules. • CONCEPT • Protein modules direct specific interactions in signal transduction pathways. • Various modules are frequently found in the same proteins

23. (III) Interaction of receptors with cytoplasmic proteins • RTKs signal via their phosphorylated tyrosine residues • The phosphotyrosines form binding sites to which subsequent signaling and scaffolding proteins bind to propagate the signal

24. Signal Transduction by the SRC Family SRC family of Protein tyrosine kinases: • 8 known members of the family: (SRC, LCK, BLK, HCK, FGR, YES, LYN, FYN ) • 60-75% amino acid identity between them (outside the unique region) • Sequences: • myristylation sequence • unique region • SH3 domain • SH2 domain • catalytic domain • regulatory region Myristylation Y Y Unique SH3 SH2 Protein kinase Membrane

25. SH2 (SRC Homology 2) domain SH2 binds to phosphotyrosine and the immediate C-terminal residues (3-5) in a sequence-specific fashion the autophosphorylated tyrosine residue in a receptor PTK binds specifically to one or more SH2-containing proteins, but may not bind to other SH2-containing proteins

26. SH2 (SRC Homology 2) domain One side of the pocket is lined with conserved basic aa and binds the phosphotyrosine The other side of the pocket is more variable and allows specific recognition of the residues at the C-terminal of the phosphotyrosine. Variations in the nature of the hydrophobic socket in different SH2 domains allow them to bind to phosphotyrosine adjacent to different sequences

27. Functions of SRC family • Potential substrates of SRC • Signal transducing proteins • many potential substrates are identified • Cytoskeletal proteins • on activation, a portion of SRC become associated with cytoskeleton. • nonactivated SRC and nontransforming mutants of v-SRC are not associated with cytoskeleton. • transformation is associated with large changes in cytoskeleton organization

28. Turning off or quenching of receptor PTK signaling • Dephosphorylation • Tyrosine phosphatases reverse the effects of Tyr phosphorylation • They are both soluble and receptor-like • Some are constitutive, most are regulated

29. P P P P P P P P P Turning off or quenching of receptor PTK signaling • Receptor internalization • endocytosis, may be autophosphorylation-mediated • Negative feedback loop by phosphorylation