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Biol403 – MAP kinase signalling

� The mitogen activated protein kinase (MAPK) pathway is asignalling cascade activated by a diverse range of effectors.

� The cascade regulates many cellular activities includingproliferation and differentiation.

� Epidermal growth factor (EGF), and a range of other growthfactors, activate MAPKs. The role of MAPKs in the EGFintracellular signalling pathway have been described in Biol220and are briefly revised here.

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� EGF binds to the EGF receptor - atyrosine kinase-based receptor.

� Binding of EGF causes the monomericreceptor to dimerize and undergocross-phosphorylation and activation.

� The SH2 domain of an adaptor protein Grb-2 (growth-factor-receptor bound protein 2) binds to phosphotyrosine residues ofthe receptor.

� Grb-2 recruits Sos (Son of Sevenless). Binding of Sos to Grb-2stimulates the GRF (guanine nucleotide releasing factor)activity of Sos.

� Sos binds to Ras and promotes a conformational change thatallows exchange of GDP for GTP in Ras.

� There are 3 isoforms of mammalian Ras – H-Ras, K-Ras and N-Ras. At their C-terminals there is a CAAX motif which can befarnesylated causing the localisation of Ras to the plasmamembrane (or other internal membranes).

� Ras possesses an intrinsic GTPase activity that eventuallyreturns the system to an inactive (GDP bound) state. Thisactivity is augmented by GAPs (GTPase activating proteins).

� Activation of Ras leads to the activation of the MAP kinasecascade.

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�The ser/thr protein kinase Raf isactivated by direct interaction withRas.

�Activated Raf phosphorylates aprotein known as MEK (MAPK/ERKkinase) at specific ser and thrresidues.

�Activated MEK1 and MEK2phosphorylate a family of proteinsknown as MAP kinases (mitogen-activated protein) or ERKs(extracellular-signal regulated proteinkinases). Significantly, thesephosphorylations occur at tyr and thrresidues i.e. MEK shows dualspecificity.

�Activated ERK1 and ERK2 migratefrom the cytosol to the nucleus, wherethey phosphorylate, and activate, avariety of transcription factorsincluding c-Jun, c-Fos and c-Myc.

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Five distinct groups of MAPKs have been identified in mammals:

The MEK/ERK cascade ( as described above);

The JNK pathway;

The p38 pathway;

ERK5 (also known as ‘big MAPK 1’ – it is twice the size of typical MAPKs) and ERK3 (an atypical MAPK) pathways.

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The JNK pathway

� The major activators of thispathway are cytokines, certain G-protein coupled receptors and cellstress. JNKs control apoptosisand development of cells of theimmune system. The c-Jun N-terminal kinase (JNK) familycontains three isoforms (JNK1,JNK2 and JNK3).

� JNK is phosphorylated by eitherMEK4 or MEK7 which arethemselves phosphorylated byseveral protein kinases includingMEKK1-4, MLK2-3 and DLK.

� Once phosphorylated JNK istranslocated to the nucleuswhere it phosphorylates severaltranscription factors including c-Jun, ATF-2, STAT3 and HSF-1.

The p38 pathway

� There are 4 members of thep38 kinase family – α, β γ and δ.

� Cytokines, hormones, G-proteincoupled receptors and cellstress (e.g. heat or osmoticshock) all stimulate thispathway.

� p38 kinases are targets forboth MEK3 and MEK6 and havemany substrates including MAPKinteracting kinases (Mnk) 1 and2 and eukaryotic initiationfactor 4e.

� The pathway regulatesangiogenesis, cell proliferation,inflammation and cytokineproduction.

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Scaffolds

� The role of scaffolds is toprovide spatial and temporalorganisation of MAPK pathways.

� Scaffolds bind multiplecomponents of the cascadebringing them into closeproximity and thereby ensuringefficient propagation ofsignalling information.

� A number of MAPK cascadescaffold proteins have now beenidentified in mammalian cells.

� These are a diverse group ofproteins, likely to have evolvedseparately, but related in thatthey are composed of multiplemodular interaction domains ormotifs.

Ste5

� One of the first scaffold proteins to be discovered was Ste5from yeast. This protein, with no apparent catalytic activity, isrequired for the mating pathway in Saccharomyces cerevisae.

� In a-type cells, the α-factor binds a G-protein coupledreceptor. Ste-5 is then recruited to the cell membrane where itbinds all three kinase components of the mating MAPK module(Ste11, Ste7, Fus3).

� Ste5 clearly promotes signalling by enforcing proximity of thesecomponents. In addition, there is allosteric activation of Fus3by Ste5 and an alternative interaction between Fus3 and Ste5makes Fus3 more susceptible to allosteric activation by Ste7.

� This latter interaction is important in that it ensures that Ste7,which also activates another MAPK (Kss1) involved in a differentpathway, activates the mating-specific Fus3 MAPK only when itis bound to Stre5.

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In response to the presence of pheromone (α-factor), Ste20 isactivated at the cell membrane and in turn activates Ste11. This initiatesa cascade of phosphorylation events leading to the activation and releaseof Fus3 from the Ste5 scaffold. By spatially coordinating the kinasesinvolved in the pheromone response, Ste5 ensures both the speed andaccuracy of signalling.

Kinase suppressor of Ras (KSR)

� KSR was originally assumed tobe a kinase; however, activityhas not been conclusivelydemonstrated and it appears,instead, to be MAPK scaffold.

� Following stimulation by growthfactors, KSR translocates fromthe cytosol to the plasmamembrane, where it forms adocking platform for cascadecomponents.

� Specifically, it binds C-Raf,MEK1/2 and ERK1/2. MEK isconstitutively bound to KSR butERK only binds in response to astimulus.

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� The proteins of this complex arepotentially important drug targets -activating mutations is Ras are foundin ~ 25% of all human tumours.

� Drugs that inhibit Raf e.g. sorafenibare used to treat renal and hepaticcarcinomas.

� Such drugs must target Raf only inthe context of the KSR-a scaffold ifthey are to have therapeuticpotential.

Sorofenib

IQGAP1

� IQGAP1 is a large, widely expressed protein that regulates manysignalling pathways. With several domains it is able to bind multipleproteins (including cytoskeletal elements) – among which arecomponents of the MAPK cascade: B-Raf, MEK1, MEK2, ERK1 andERK2.

� Although the association between ERK and IQGAP1 is constitutive, thebinding of MEK1 only occurs in the presence of stimulus (EGF) whereasthe binding of MEK2 is inhibited in the presence of EGF. This mayprovide an explanation for the observation that MEK1 promotescellular proliferation whereas MEK2 promotes cellular differentiation.

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ββββ-arrestins

β-arrestins are well established regulatorsof G-protein coupled receptors (causingdissociation of heterotrimeric G proteins).They also directly regulate MAPKcascades. In response to activation ofprotease activated receptor (PAR), β-arrestin recruits RAF, MEK and ERK tothe receptor, enhancing activation of ERK.The complexes accompany the receptor toearly endosomes thereby preventing thetranslocation of active ERK to thenucleus, restricting its activity tocytosolic substrates.

Similar expression to FGF (Sef)

Sef apparently captures MEK/ERK complexes at the Golgi and inhibitsnuclear localisation of ERK, restricting ERK activity to cytosolic substrates(Sef is actually interleukin-17 receptor D).

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� Summary of the roleof MAPK scaffoldproteins in thespatial and temporalorganisation of thesignalling cascade.

� Rapid ERK activationvia PK-C or Src istransient and allowsERK translocation tothe nucleus.

� Sustained ERKactivation is via anendosomal β-arrestindependent processthat confinessignalling to thecytosol.

Scaffolds and MAPK specificity

� An aspect of MAPK signalling that is poorly understood ishow a particular stimulus elicits the correct response (i.e.MAPK specificity). Given the diverse range of cellularresponses induced by numerous different activators, all ofwhich signal through the MEK/ERK pathway, this is asignificant issue.

� Scaffolds are able to control many aspects of MAPKsignalling. By bringing together individual components of thecascade, scaffolds facilitate their interactions andpropagation of the signal. The scaffold also effectively‘insulate’ these components from other signalling pathwaysi.e. scaffolds can lead to preferential activation andinhibition of signalling cascades.

� Scaffolds that interact with, or are regulated by, differentsignalling pathways will allow ‘cross-talk’ between signallingcascades.

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Summary

� The mitogen activated protein kinase (MAPK) pathway is asignalling cascade that regulates many cellular activitiesincluding proliferation and differentiation.

� Five distinct groups of MAPKs have been identified in mammals– the MEK/ERK cascade, the JNK pathway, the p38 pathwayand ERK5/ERK3 pathways.

� Protein scaffolds bind multiple components of the cascadesbringing them into close proximity and thereby ensuringefficient propagation of signalling information.

� Scaffolds provide spatial and temporal organisation of MAPKpathways and provide an explanation for the specificity ofMAPK signalling pathways.

References

Brown, M. D. & Sacks, D. B. (2009) Cellular Signalling 21, 462-469.– protein scaffolds in MAPK signalling.

Alexa, A et al., (2010) FEBS J. 277, 4367-4382. – Scaffoldproteins.

Wimmer, R & Baccarini, M. (2010) Trends Biochem. Sci. 35, 660-668. - protein interactions in MAPK signalling.

Kiel, C., & Serrano, L. (2012) Curr. Opin. Biotech. 23, 305-314 MAPK signalling.

Drew, B. A. et al., (2012) Biochim. Biophys. Acta 1825, 37-48 -MEK5/ERK5 pathway.