Cell Signaling II Signal Transduction pathways Cell Biology Lecture 13

Readings and Objectives Russell Chapter 8 (not sufficient) Cooper: Chapter 15 Topics Lecture 12 Signaling Molecules and Their Receptors Functions of Cell Surface Receptors Lecture 13 Pathways of Intracellular Signal Transduction Signal Transduction and the Cytoskeleton Signaling Networks

Intracellular Signal Transduction Pathways Intracellular signal transduction- chain of reactions, transmits signals/cell surfaceintracellular targets First studied for epinephrine Signals glycogen breakdown to glucose Earl Sutherland (1958): action of epinephrine was mediated by an increase in cyclic AMP (cAMP) Concept: cAMP is a second messenger Noble prize 1971 1915-1974

cAMP Signal Transduction Pathways PKA activation Epinephrine receptor coupled to adenylyl cyclase via a G protein increasing the concentration of cAMP cAMP signaling & cell responses Metabolic regulation Cytosolic Protein Kinase A activation (PKA) tetramer of regulatory and catalytic subunits, ie R2C2 (inactive) cAMP binding of “R” dissociation of catalytic subunits (active) A serine/threonine kinaseActivation or inactivation of substrate proteins

cAMP Signal Transduction Pathways Phosphorylation of two downstream enzymes: Glycogen synthase inactivated glycogen synthesis↓ Phosphorylase kinase activated phosphorylates Glycogen phosphorylase (active) Glu-1P↑

cAMP Signal Transduction Pathways 2. Gene regulation Increased cAMP activate transcription Free PKA C-subunit translocated to the nucleus binds Genes containing a regulatory sequence—the cAMP response element, or CRE phosphorylates the transcription factor CREB (CRE-binding protein). Recruits RNA polymerase expression of cAMP-inducible genes Proliferation, differentiation, memory, cognition Review article: Transcriptional regulation by cAMP

cAMP Signal Transduction Pathways Protein phosphorylation is reversed by protein phosphatases terminates responses initiated by receptor activation of protein kinase

Secondary messenger DAG and IP3 signaling PLC-γ binds receptor protein tyrosine kinases via SH2 domain phosphorylated (active) PLC- γ stimulates hydrolysis of PIP2 to DAG and IP3 (how?) DAG and IP3 are secondary messengers IP3 regulates Ca2+ DAG activates PKC family PLC=Phospholipase C PIP2: Phosphatidylinositol 4,5-bisphosphate IP3: Inositol 1,4,5-trisphosphate DAG: Diaceyl glycerol

Secondary messenger DAG and IP3 signaling PLC-γ binds receptor protein tyrosine kinases via SH2 domain phosphorylated (active) PLC- γ stimulates hydrolysis of PIP2 to DAG and IP3 (how?) DAG and IP3 are secondary messengers IP3 regulates Ca2+ DAG activates PKC family PLC=Phospholipase C PIP2: Phosphatidylinositol 4,5-bisphosphate IP3: Inositol 1,4,5-trisphosphate DAG: Diaceyl glycerol

Secondary messenger DAG and IP3 signaling DAG remains associated with the plasma membrane and activates protein-serine/threonine kinases of the protein kinase C family. IP3 , a small polar molecule, released to the cytosol Stimulates release of Ca2+ from the ER by binding to receptors that are ligand-gated Ca2+ channels

Secondary messenger DAG and IP3 signaling Calmodulin is activated when Ca2+ concentration increases CaM kinase family are activated by Ca2+/calmodulin they phosphorylate and activate other proteins such as, protein kinases, phosphatases, metabolic enzymes, ion channels, and transcription factors (eg CREB) Also regulates synthesis and release of neurotransmitters

Secondary messenger DAG and IP3 signaling nonmuscle cells and smooth muscles, contraction is regulated by phosphorylation of myosin light chain catalyzed by myosin light chain kinase, which is regulated by the Ca2+ binding protein calmodulin

Secondary messenger DAG and IP3 signaling Increased [Ca2+ ] signals further release of Ca2+ from the ER by opening Ca2+ channels (ryanodine receptors) in the ER membrane. Ca2+ is a versatile second messenger that controls a wide range of cellular processes These pathways function coordinately to regulate many cellular responses

PI 3/Akt signaling pathway PIP2 is also the start of another signaling pathway PIP2 is phosphorylated by phosphatidylinositide (PI) 3-kinase This yields a second messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP3)

PI3/Akt signaling pathway PIP3 targets a protein-serine/threonine kinase called Akt and also binds protein kinase PDK1 Activation of Akt also requires protein kinase mTOR (in a complex called mTORC2) which is also stimulated by growth factor mTOR: mammalian target of rapamycin PDK1: phosphoinositide dependent protein kinase-1 GSK3: glycogen synthase kinase 3 Bad: Bcl2 associated death promoter (promotes apoptosis)

PI3/Akt signaling pathway Akt phosphorylates several target proteins, transcription factors, and other protein kinases Transcription factors include members of the Forkhead or FOXO family If growth factors are not present, Akt is not active FOXO travels to the nucleus, stimulates transcription of genes that inhibit cell proliferation, or induce cell death

PI3/Akt signaling pathway When growth factors attached to receptor/tyrosine kinases Akt is phosphorylated (active) Akt phosphorylation of FOXO sequesters it in inactive form Akt inhibits GSK-3, the general inhibitor of translation Inhibition of GSK-3 relieves translation Cells are prepared to proliferate

MAP Kinase Signaling Pathways MAP kinases (mitogen-activated protein kinases) are protein-serine/threonine kinases Conserved across eukaryotic cells; three groups of MAP kinases

ERK Signaling Pathway ERK (extracellular signal-regulated kinase) family, first to be identified in MAPKs regulation of meiosis, mitosis, cell proliferation and differentiation Ligands: growth factors, cytokines and viral infection, carcinogenic chemicals

ERK Signaling Pathway ERK activation mediated by Ras, Raf, MEK kinase cascade Activation of Rasactivation of Raf protein serine/threonine kinase Raf phosphorylates and activates a second protein kinase called MEK (MAPK/ERK Kinase) MEK activates ERK transcriptional activation

ERK Signaling Pathway Ras: guanine nucleotide-binding protein that function like α subunits of G proteins Ras is activated by guanine nucleotide exchange factors (GEF) Sos=specific GEF for Ras GTPase-activating proteines GTP hydrolysis Ras-GDP becomes inactive

ERK Signaling Pathway Grb2: SH2 domain containing protein associated with Sos RPTK activation by ligand recruits Grb2/Sos to membrane Grb2/Sos contacts Ras-GDP GTP replaces GDP in Ras Ras-GTP activated and phosphorylates Raf Raf initiates a protein kinase cascade ERK activation

ERK Signaling Pathway ERK goes to the nucleus, phosphorylates Elk-1 transcriptional induction of immediate-early genes (~ 100 genes) serum response element (SRE), recognized by transcription factors serum response factor (SRF) and Elk-1 immediate-early genes encode transcription factors Activate downstream genes called secondary response genes Cell proliferation and growth

ERK Signaling Pathway Specificity of MAP kinase signaling is maintained in part by their physical association on scaffold proteins For example, the KSR scaffold protein organizes ERK and its upstream activators Raf and MEK into a signaling cassette

JAK/STAT Pathway Direct signaling from receptor to nucleus Ligand: cytokines Receptors: Janus Kinases (JAK), nonreceptor protein-tyrosine kinase STAT: Signal Tansducer & Activators of Transcription Transcription factors, contain SH2 domains that mediate binding to phosphotyrosine sequences STATs activated, dimerized, translocate to nucleus Activate transcription

Notch Pathway direct cell-cell interactions during development Notch a receptor for signaling by transmembrane proteins (e.g., Delta) on adjacent cells Ligand binding proteolytic cleavage of cytosolic domain of Notch translocated into the nucleus converts a transcription factor (CSL in mammals) from a repressor to an activator Downstream genes code for other transcriptional factors Cell developmental differentiation Minireview: Notch signaling

Integrins and Signal Transduction binding of integrins to the extracellular activation of FAK ( focal adhesion kinase), a nonreceptor protein-tyrosine kinase provides binding sites for Grb2-Sos complex, leading to activation of Ras/ERK, PI 3-kinase

Integrins and Signal Transduction Rho subfamily of small GTP-binding proteins (Rho, Rac, and Cdc42) regulate organization of the actin cytoskeleton Rho family proteins promote actin polymerization