Cell Signaling II Cell Signaling II Signal Transduction pathways Cell Biology Lecture 13.

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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