Regulation of activity C-Raf

artist s impression of autoinhibited state of c-raf, reinforced associated 14-3-3 protein dimers, bound phosphorylated twin motifs.

as mentioned above, regulation of c-raf activity complex. gatekeeper of erk1/2 pathway, kept in check multitude of inhibitory mechanisms, , cannot activated in single step. important regulatory mechanism involves direct, physical association of n-terminal autoinhibitory block kinase domain of c-raf. results in occlusion of catalytic site , full shutdown of kinase activity. closed state can relieved if autoinhibitory domain of raf engages partner competing own kinase domain, importantly gtp-bound ras. activated small g-proteins can break intramolacular interactions: results in conformational change ( opening ) of c-raf necessary kinase activation , substrate binding.

14-3-3 proteins contribute autoinhibition. 14-3-3 proteins known form constitutive dimers, assemblies have 2 binding sites. dimer acts molecular handcuff , locking binding partners @ fixed distance , orientation. when precisely positioned twin 14-3-3 binding motifs engaged single 14-3-3 protein dimer (such 14-3-3 zeta), become locked conformation promotes autoinhibition , not allow disengagement of autoinhibitory , catalytic domains. lockdown of c-raf (and other rafs ksrs) controlled motif phosphorylation. unphosphorylated 14-3-3 associating motifs not bind partners: need phosphorylated on conserved serines (ser 259 , ser 621) first, other protein kinases. important kinase implicated in event tgf-beta activated kinase 1 (tak1), , enzymes dedicated removal of these phosphates protein phosphatase 1 (pp1) , protein phosphatase 2a (pp2a) complexes.

note 14-3-3 binding of raf enzymes not inhibitory: once raf open , dimerizes, 14-3-3s can bind in trans, bridging 2 kinases , handcuffing them reinforce dimer, instead of keeping them away each other. further modes of 14-3-3 interactions c-raf exist, role not known.

dimerisation important mechanism c-raf activity regulation , required raf activation loop phosphorylation. normally, open kinase domains participate in dimerisation. unlike b-raf, readily forms homodimers itself, c-raf prefers heterodimerisation either b-raf or ksr1. homodimers , heterodimers behave similarly. b-raf homodimer kinase domain structure shows activation loops (that control catalytic activity of known protein kinases) positioned in active-like conformation in dimer. due allosteric effect of other molecule binding side of kinase; such dimers symmetric , have two, partially active catalytic sites. @ stage, activity of raf kinases low, , unstable.

the activation cycle of mammalian raf proteins, exemplified b-raf (a simplified overview, not showing steps).

to achieve full activity , stabilize active state, activation loop of c-raf needs phosphorylated. kinases known perform act raf family kinases themselves. other kinases, such pak1 can phosphorylate other residues near kinase domain of c-raf: precise role of these auxiliary kinases unknown. in context of c-raf, both c-raf , ksr1 needed transphosphorylation step. due architecture of dimers, phosphorylation can take place in trans (i.e. 1 dimer phosphorylates another, in four-membered transitional complex). interacting conserved arg , lys residues in kinase domain, phosphorylated activation loops shift conformation , become ordered, permanently locking kinase domain active state until dephosphorylated. phosphorylated activation loops render kinase insensitive presence of autoinhibitory domain. ksrs cannot undergo last step miss phosphorylatable residues in activation loops. once c-raf activated, there no further need so: active raf enzymes can engage substrates. protein kinases, c-raf has multiple substrates. bad (bcl2-atagonist of cell death) directly phosphorylated c-raf, along several types of adenylate cyclases, myosin phosphatase (mypt), cardiac muscle troponin t (tntc), etc. retinoblastoma protein (prb) , cdc25 phosphatase suggested possible substrates.

the important targets of raf enzymes mkk1(mek1) , mkk2(mek2). although structure of enzyme-substrate complex c-raf:mkk1 unknown, can precisely modelled after ksr2:mkk1 complex. here no actual catalysis takes place, thought highly similar way raf binds substrates. main interaction interface provided c-terminal lobes of both kinase domains; large, disordered, proline-rich loop unique mkk1 , mkk2 plays important role in positioning raf (and ksr). these mkks become phosphorylated @ at least 2 sites on activation loops upon binding raf: activate them too. targets of kinase cascade erk1 , erk2, selectively activated mkk1 or mkk2. erks have numerous substrates in cells; capable of translocating nucleus activate nuclear transcription factors. activated erks pleiotropic effectors of cell physiology , play important role in control of gene expression involved in cell division cycle, cell migration, inhibition of apoptosis, , cell differentiation.