1 / 33

Signal Transduction

Signal Transduction. Biochemistry – February 23, 2005 Chapter 12 – parts 12.3, 12.4. Signaling Characteristics. Types of Receptors. Receptor tyrosine kinases (RTKs). ligand-binding domain, single transmembrane domain, conserved intracellular domain

chance
Download Presentation

Signal Transduction

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Signal Transduction Biochemistry – February 23, 2005 Chapter 12 – parts 12.3, 12.4

  2. Signaling Characteristics

  3. Types of Receptors

  4. Receptor tyrosine kinases (RTKs) • ligand-binding domain, single transmembrane domain, conserved intracellular domain • typical ligands are peptide hormones and growth factors – all will stimulate cell division • Insulin - released by pancreasin the presence of high blood[glucose] • Vascular endothelium growth factor • Insulin-like growth factor • Platelet-derived growth factor • Epidermal growth factor

  5. Receptor tyrosine kinases (RTKs) • Ligand binding causes dimerization, cross-phosphorylation on tyrosine • Phosphorylated tyrosines shift position of activation loop, exposing substrate binding site – making it active. • Regulatory mechanism is seen in many protein kinases

  6. Insulin Receptor Tyrosine Kinase activity • Insulin receptor phosphorylates Insulin receptor substrate-1 (IRS-1) on specific tyrosine residues

  7. SH2 domains • Phosphotyrosine recognized by SH2 (src-homology) domain found on many signaling proteins • non-receptor tyrosine kinases (src) • Phospholipase C-  (PLC-), • GTPase activating proteins (GAPs) • Phosphoinositide-3 kinase (PI-3K) • Adapter proteins such as Grb2

  8. Response to Insulin • Phosphorylated IRS-1 bound by Grb2, which binds Sos (son of sevenless) • Sos is a guanine nucleotide exchange factor (GEF), activates ras by triggering release of GDP, binding of GTP by ras • Ras is a member of the small GTPase family – myristylation associates ras with membrane

  9. ras signaling • activated ras binds raf, a S/T kinase, localizing it to the plasma membrane • raf phosphorylates MEK (MAP and ERK kinase) (Y/T kinase), • MEK phosphorylates ERK & MAP kinase (S/T kinase) • ERK & MAP kinase phosphorylate many different proteins involved in cell division and response to insulin • Transcription factors • Cell cycle regulators such as Cyclin dependent kinases (cdk’s)

  10. Insulin response • Phosphorylated IRS-1 activates PI-3K • PI-3K phosphorylates PIP2 to form PIP3 • PIP3 activates PDK1 which activates protein kinase B (PKB) • PKB phosphorylates Glycogen Synthase Kinase 3 (GSK3) to INACTIVATE it, preventing phosphorylation of Glycogen Synthase (GS) • Phosphatases can dephosphorylateGS to activate it • GS uses available glucose to produce glycogen, a storage formof glucose. • End result – high blood [glucose] leads to glycogen synthesis

  11. Stop here!

  12. 7TM receptors • Receptors bind extracellular ligand, such as epinephrine, triggering conformational change. Change allows interaction with “downstream effectors” – often a G-protein. • Receptors often referred to as GPCR’s – G-protein coupled receptors.

  13. G-proteins • Activated receptor functions as GEF – guanine nucleotide exchange factor • GDP released by heterotrimeric G-protein, GTP bound by a subunit, conformational changecauses dissociation from bg subunits. • Activated (GTP bound) a-subunit interacts with downstream effectors

  14. b-adrenergic receptor stimulates Gs • Activated Gsa activates Adenylate cyclase which produces cAMP • Adenylate cyclase acts as a GAP – GTPase activating protein • Hydrolysis of GTP inactivates Gsa subunit

  15. cAMP activates protein kinase A • cAMP activates protein kinase A by binding to regulatory subunit, which then releases active catalytic subunit • PKA is a S/T kinase that phosphorylates many proteins, triggering a variety of responses • metabolic changes • changes in gene expression • changes in ion transport

  16. Response to hormones • Epinephrine (adrenalin) is the fight or flight hormone - energy reserves must be mobilized in preparation for “action” • Glucagon is produced by pancreas in response to low blood [glucose] • Liver and muscle respond by making energy reserves available

  17. Turning off response • Receptor turned off by phosphorylation on intracellular domain and binding of b-arrestin • result is that more hormone binding to other receptors is required for response.

  18. Phospholipase C (PLC) • Some 7TM receptors activate specific G-proteins that activate phospholipase C (PLC). • PLC cleaves PIP2 (phosphatidyl inositol-4,5-bisphosphate) present in the cytosolic leaflet of the plasma membrane) to IP3 (inositol trisphosphate) and DAG (diacylglycerol).

  19. Phospholipase C • PH and C2 domains bind lipids, associate protein with membrane • EF hand domains bind Calcium • Catalytic domain catalyzes reaction PIP2 IP3 + DAG • Regulatory domain interacts with activator

  20. Inositol Lipid Signalling • IP3 diffuses to ER membrane, binds ion channel receptor, causing release of calcium • calcium triggers exocytosis, smooth muscle contraction • calcium is bound by calmodulin, which activates cAMP phosphodiesterase (turning off cAMP pathway) and specific protein kinases

  21. Protein Kinase C • calcium binds C2 domain of protein kinase C - causes association with membrane • DAG binds C1 domains of protein kinase C, removing pseudosubstrate from active site • PKC phosphorylates specific proteins to cause a cellular response • DAG is also a precursor of arachidonic acid and prostaglandins

  22. Calcium signaling • Can alter and monitor levels • A23187 - Ca ionophore • EGTA - Ca chelator • fura-2, fluo-3, aequorin - Ca sensitive fluorescent dyes, proteins

  23. Calcium effectors • Calmodulin binds 4 Ca ions • Ca-Calmodulin binds basic amphipathic helices on target proteins, triggering conformational changes that activate the protein • Ca-ATPase to restore Ca levels • Calmodulin dependent protein kinase II (CaM kinase II) - different targets in different cell types

  24. G-protein families

  25. Growth Factor Signalling • often SH2 proteins also have SH3 domain (drk-Grb2 adaptors)- involved in interactions with other proteins, such as Guanine nucleotide exchange factors (e.g. sos) • GTP binding activates ras (a small monomeric GTPase) whichactivates a protein kinase cascade.

  26. ras pathway • ras pathway involved in many growth and development pathways • Drosophila R7 photoreceptor pathway • mutants lack R7 cell – sevenless • sos = son of sevenless • boss = bride of sevenless

  27. Adapter Proteins • SH2 and SH3 domains of different proteins have different specificities for target proteins based on sequence surrounding phosphotyrosine (SH2) or conserved prolines (SH3)

  28. ras activation • GRB2 (adaptor protein)interacts with RTK and Sos (son of sevenless)-likeprotein • GEF activity of Sos protein causes ras to release GDP, bind GTP and become activated. • ras involved in cell growth/development signalling pathways • constitutive ras mutations found in up to 50% of human cancers

  29. ras signaling • 14-3-3 proteins inhibit raf activity, ras causes the proteins to dissociate • Ksr required for proper interaction between raf, 14-3-3, MEK, MAP kinase.

  30. MAP kinase pathway evolution • single-celled eukaryotes do not depend on growth signals from other cells but do use MAP kinases to respond to major changes in environment • Scaffold proteins homologous to ksr link specific effector kinases to signal perception • specificity of response dependent upon specificbinding of effectors.

  31. Transcriptional activation • In response to growth factor induction of MAP kinase, pp90rsk and MAP kinase activate serum response factors, which induce expression of fos and jun. • fos/jun complex (AP-1) activates expression of genes necessary for progression through cell cycle.

  32. Growth Hormone Receptor • Hormone binding causes dimerization • dimerized receptor phosphorylated by JAK (Janus Kinase) • JAK SH2 domains bind phosphotyrosine on receptor • JAK phosphorylates target proteins (e.g STATs) • STATs activate transcription

  33. Signal Amplification • Signal transduction cascades amplify a signal • ligand-receptor complex can activate many G • each G can activate adenyl cyclase such that many cAMP are produced • each cAMP activates cAPK which can phosphorylate multiple proteins • each protein can then affect multiple downstream effectors

More Related