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 typical ligands are peptide hormones and growth factors – all will stimulate cell division –Insulin - released by pancreas in the presence of high blood [glucose] –Vascular endothelium growth factor –Insulin-like growth factor –Platelet-derived growth factor –Epidermal growth factor

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

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

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

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

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)

Insulin response Phosphorylated IRS-1 activates PI-3K PI-3K phosphorylates PIP 2 to form PIP 3 PIP 3 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 dephosphorylate GS to activate it GS uses available glucose to produce glycogen, a storage form of glucose. End result – high blood [glucose] leads to glycogen synthesis

Stop here!

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.

G-proteins Activated receptor functions as GEF – guanine nucleotide exchange factor GDP released by heterotrimeric G- protein, GTP bound by  subunit, conformational change causes dissociation from  subunits. Activated (GTP bound)  subunit interacts with downstream effectors

 -adrenergic receptor stimulates Gs Activated Gs  activates Adenylate cyclase which produces cAMP Adenylate cyclase acts as a GAP – GTPase activating protein –Hydrolysis of GTP inactivates Gs  subunit

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

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

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

Phospholipase C (PLC) Some 7TM receptors activate specific G-proteins that activate phospholipase C  (PLC  ). PLC cleaves PIP 2 (phosphatidyl inositol-4,5-bisphosphate) present in the cytosolic leaflet of the plasma membrane) to IP 3 (inositol trisphosphate) and DAG (diacylglycerol).

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

IP 3 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 Inositol Lipid Signalling

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

Calcium signaling Can alter and monitor levels –A Ca ionophore –EGTA - Ca chelator –fura-2, fluo-3, aequorin - Ca sensitive fluorescent dyes, proteins

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

G-protein families

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) which activates a protein kinase cascade.

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

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)

ras activation GRB2 (adaptor protein) interacts with RTK and Sos (son of sevenless)-like protein 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

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

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 specific binding of effectors.

Transcriptional activation In response to growth factor induction of MAP kinase, pp90 rsk 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.

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

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