Cell Signaling Cell Signaling IIIISignal Transduction pathways

Cell BiologyCell BiologyLecture 13Lecture 13

Readings and ObjectivesReadings and Objectives• ReadingReading

– Russell Chapter 8 (not sufficient)

– Cooper: Chapter 15• TopicsTopicsLecture 12• Signaling Molecules and Their Receptors • Functions of Cell Surface ReceptorsLecture 13• Pathways of Intracellular Signal Transduction• Signal Transduction and the Cytoskeleton• Signaling Networks

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Intracellular Signal Transduction PathwaysIntracellular Signal Transduction Pathways• Intracellular signal transduction- chain of reactions,

transmits signals/cell surfaceintracellular 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

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1915-19741915-1974

cAMP Signal Transduction PathwayscAMP Signal Transduction Pathways

• Epinephrine receptor coupled to adenylyl cyclase via a G protein increasing the concentration of cAMP

cAMP signaling & cell responsescAMP signaling & cell responses1.1. Metabolic regulationMetabolic 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 kinaseActivation or

inactivation of substrate proteins

4PKA activation

cAMP Signal Transduction PathwayscAMP Signal Transduction PathwaysPhosphorylation of two downstream enzymes:• Glycogen synthase inactivated glycogen synthesis↓

• Phosphorylase kinase activated phosphorylates Glycogen phosphorylase (active) Glu-1P↑

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cAMP Signal Transduction PathwayscAMP Signal Transduction Pathways2. Gene regulation2. 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 6

cAMP Signal Transduction PathwayscAMP Signal Transduction Pathways

• Protein phosphorylation is reversed by protein phosphatases

• terminates responses initiated by receptor activation of protein kinase

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Secondary messenger DAG and IP3 signalingSecondary 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

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PLC=Phospholipase CPIP2: Phosphatidylinositol 4,5-bisphosphate IP3: Inositol 1,4,5-trisphosphateDAG: Diaceyl glycerol

Secondary messenger DAG and IP3 signalingSecondary 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

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PLC=Phospholipase CPIP2: Phosphatidylinositol 4,5-bisphosphate IP3: Inositol 1,4,5-trisphosphateDAG: Diaceyl glycerol

Secondary messenger DAG and IP3 signalingSecondary 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 10

Secondary messenger DAG and IP3 signalingSecondary 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

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Secondary messenger DAG and IP3 signalingSecondary 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

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Secondary messenger DAG and IP3 signalingSecondary 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

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• 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)

PI 3/Akt signaling pathway

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• 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

PI3/Akt signaling pathway

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mTOR: mammalian target of rapamycinPDK1: phosphoinositide dependent protein kinase-1GSK3: glycogen synthase kinase 3Bad: Bcl2 associated death promoter (promotes apoptosis)

• 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

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• 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

PI3/Akt signaling pathway

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• MAP kinases (mitogen-activated protein kinases) are protein-serine/threonine kinases

• Conserved across eukaryotic cells; three groups of MAP kinases

MAP Kinase Signaling Pathways

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• ERK (extracellular signal-regulated kinase) family, first to be identified in MAPKs

• regulation of meiosis, mitosis, cell proliferation and differentiation• Ligands:Ligands: growth factors, cytokines and viral infection, carcinogenic

chemicals

ERK Signaling Pathway

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• ERK activationERK activation mediated by Ras, Raf, MEK kinase cascade• Activation of Rasactivation 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

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• RasRas:: 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

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• Grb2: 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

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• 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

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• 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

ERK Signaling Pathway

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• 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

JAK/STAT PathwayJAK/STAT Pathway

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• 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

Notch PathwayNotch Pathway

26Minireview: Notch signaling

• 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 TransductionIntegrins and Signal Transduction

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• Rho subfamily of small GTP-binding proteins (Rho, Rac, and Cdc42) regulate organization of the actin cytoskeleton

• Rho family proteins promote actin polymerization

Integrins and Signal TransductionIntegrins and Signal Transduction

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