Outline

• Pathway review
• Crosstalk
• Modulation

Pathway review: lets go over the key pathways in a manner organized by the downstream kinases (see Fig)

• MAPK: multiple per cell, all activated by a kinase cascade w/ Ras and RTK upstream
• peptide->RTK dimer->adaptor GRB2(SH2,SH3)->SOS(GEF)->RasGTP->Raf->MEK-MAPK
• PKA: cAMP (2^nd messenger dep.), upstream activator is trimeric G & GPCR acting on Ad. Cyc.
• catechol->7TMD GPCR->GsGTP->Ad.Cyc.->cAMP->PKA
• PKC: activated by Ca & DAG which come from PLC activation by either RTK or GPCR
• peptide->RTK->adaptor->PLC->DAG & IP3->IP3R->Ca->PKC
• catechol->7TMD GPCR->GoGTP->PLC->DAG & IP3->IP3R->Ca->PKC
• PKB: activated by binding PI-3,4,5 via PH domain
• peptide->RTK->adaptor (SH2,P-tyr)->PI3K(SH2)->PI3,4,5->PKB(PH)
• CaMK: activated by calmodulin in Ca bound form, Ca comes from ER (IP3R) or PM Ca channels
• voltage or ligand->PM Ca++ channel->Ca++->CaM->CaMK
• peptide->RTK->adaptor->PLC->DAG & IP3->IP3R->Ca->CaM->CaMK
• catechol->7TMD GPCR->GoGTP->PLC->DAG & IP3->IP3R->Ca->CaM->CaMK
• substrates of these kinases are gene regulatory proteins and "target proteins"
• specificity of these kinases for pathway specific substrates comes from:
• pathway specific isoforms of the kinases (e.g. MAPKs)
• restricted subcellular localization of the kinase & its appropriate substrates "signaling complex"
• tissue specific substrate expression, ie only appropriate substrates are expressed in a given tissue

Crosstalk

• The above paths are presented as basically linear w/ some overlap
• Actually there is considerable crosstalk between the paths that allows the cell interpret complex cues
• Some of the important crosstalk concepts are:
• divergence (distribution):
• one signal activates coordinated set of responses
• within a divergent response can be:
  activation of other signaling molecules (2nd messengers, adaptors, effectors)
  inhibition of other signaling molecules ie "If A then inhibit B"
• convergence:
• multiple signals activate pathways that usually converge
• to the extent that they don’t converge the correspondance between the
  "signal mixture" and the response it generates would be an outcome of
  the specific substrates that are phosphorylated
• convergence can yield important outcomes:
• "OR" type logic (this is basically summation)
• the response strength/duration is governed by the amount of signal
• A or B can activate, A+B will usually be greater than A or B alone
• mechanisms are many & straightforward:
  ie 1 path activates Adenylate cyclase-> more cAMP
  the other path inhibits phosphodiesterase-> more cAMP
  or different 2nd messengers trigger same response
• "AND" type logic (integration):
• the response requires both paths to be activated, either alone is ineffective
• mechanism can be:
  2 required activating phosph sites hit by distinct kinases in 2 paths
  dimer formation where monomers are activated by 2 paths

Modulation

• Regulatory Feedback can occur at multiple levels & adjusts the cells responsiveness
• an example is receptor inactivation in response to ligand
• 2 types of GPCR desensitization both involving receptor cyto tail phosphorylation
• heterologous desensitization (note that this is a form of "if A inhibit B")
• activated A Kinase phosphorylates cyto tail of all GPCRs
• the phospho-receptors become bound by arrestin
• arrestin bound Rs can no longer interact with the trimeric G
• homologous desensitization
• ligand binding makes receptor a substrate for BARK (beta-adrenergic R kinase)
• BARK only phosph beta-adrenergic receptor thus arrestin only binds & inhibits it
• these feedback desensitization mechs allow cells to maintain responsiveness to additional ligand
• in other words the % inactivated receptors mirrors the constant ligand concentration
• thus short term changes above this level can still lead to activation
• Receptor downregulation by endocytosis
• this is the principle mechanism for growth factor receptors
• this is important in cancer because of mutations that prevent downregulation-> growth
• the net effect is that ligand binding stimulates endocytosis (ie sorting into CCVs)
• mechanism for EGF is that EGFR dimerization exposes adaptin binding site (sorting signal)
• EGFR is inefficiently recycled (approx.50%) so it is degraded in lysosomes
• degradation is a sensitive way to regulate cells resposiveness
• assume 1000 occupied receptors/cell for response, and Kd of 10^-8
• if cell has 10,000 R => 1x10^-9M EGF for response
• if cell has 2,000 R => 1x10^-8M EGF for response (10X more ligand)
• if cell has 1,200 R => 5x10^-8M EGF for response (50X more ligand)
• interesting to note that arrestin also binds clathrin
• -> internalization of beta-adrenergic Receptor as well as steric blocking of G association