Signal Transduction

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