Outline

• MAP kinases
• 2nd Messengers

MAP kinases

• multiple MAPK pathways (even in single cells) regulate diverse processes
• distinct receptors (w/ distinct ligands) serve to activate specific paths
• not just RTKs, but GPCRs, ion channels etc.
• each pathway may have specific MAP kinases
• large family of conserved kinases w/ differing substrate specificities
• but each pathway usually shares upstream components (raf, MEK)
• activation of the MAPKs is via a common kinase cascade
• resulting in phosphorylation at the phosphorylation lip
• so, where does the specificity come from
• specificity comes from assembly into large pathway specific signaling complexes
• scaffold proteins! bind each component in the signaling pathway
• the scaffold protein may be the MEK or it may not be a kinase at all
• affinity of a given MAPK for the scaffold pathway is the key
• if the MAPK is deleted, another may bind & then specificity is lost
• in Fus3 delete (mating MAPK), mating factor stimulates mating & filamentation
• this is because Kss1 (filamentation MAPK) binds mating scaffold
• in kinase dead Fus3 mutants, the Fus3 still binds mating scaffold
  ->no response to mating, but specificity maintained

2^nd Messengers

• play a key role in most of the important aspects of signal transduction
• e.g. transfer, transformation, amplification, distribution, and modulation

cAMP

• many molecules generated by activated Ad. Cyclase in PM (amplification)
• these can diffuse to a distant site (transfer) to activate cAMP-dep kinase (transformation)
• multiple targets are usually activated (distribution) & and these may serve dual regulatory function
• cAMP binds:
• regulatory subunits or regulatory domains of A kinases
• these inhibitors usually act as pseudo substrates blocking active site
• cAMP activation coordinates multiple effects
• e.g. A Kinase tetramer: cooperative binding to regulatory subunits, conformational change -> displaced reg subunits, leaving active A kinase dimer
• one function of active A kinase is activation of GPK (glycogen phosphorylase kinase)
  -> active GPK phosphorylates & activates GP (glycogen phosphorylase)
  -> GP breaks glycogen into glucose-1-P
• another function of A kinase is phosphorylation & inactivation of GS (glycogen synthase)->thus glucose is not converted into glycogen
• finally A kinase also phosphorylates IP (inhibitor of phosphoproteinphosphatase)
  ->phospho-IP binds & blocks PP (phosphoprotein phosphatase)
  ->thus all the phospho forms are maintained until cAMP decreases
• A kinase anchoring proteins (AKAPs) can produce region specific cAMP responses
• e.g. changes at leading edge in motile cells specifically respond to cAMP
• in part because A kinase can only interact w/ substrates at this position

Phosphatidyl Inositol (PI) Signaling

• signaling pathways (eg RTKs or GPCRs) alter lipid kinase, phosphatase & lipase activities
• these enzymes modify PI to generate second messengers of three types
• PM associated phosphoinositides: these are then bound by adaptors (e.g PH or FYVE)
• PM associated DAG: this will binds & activate membrane associated PKC
• soluble (cytoplasmic) IP3: this binds the IP3 receptor & Ca channel in the ER
• Ca release into cyto causes PKC recruitment at PM
• PKC phosphorylates many substrates regulating diverse processes
• The pathway is:

DAG+IP-1	DAG+IP-1,4	DAG+IP-1,4,5
PI	PI-4 (aka PIP)	PI-4,5 (aka PIP2)
PI-3	PI-3,4	PI-3,4,5

• IP3 receptor is tetrameric multi TMD Calcium channel w/ large IP3 binding cyto domain
• IP3 binding opens calcium channel
• channel more sensitive to IP3 if cyto calcium is already elevated-> feed forward
• depletion of ER storage pool of Ca-> signal from IP3R to PM store-operated Ca channel
• Ca influx at PM increase cyto Ca which is then taken back into ER by Ca ATPase

Ca++

• in addition to translocation of PKC, Ca++ activates effector calmodulin
• calmodulin binds 4 Ca and undergoes a profound conformational change
• calcium bound calmodulin binds and activates:
• calcium-calmodulin dependent protein kinases (CaMK) to elicit transcription etc.
• cAMP phosphodiesterase to turn off a cAMP pathway

cGMP, NO

• cGMP activates cGMP dep kinases similar to cAMP
• it is synthesized by a PM enzyme linked receptor or by a
• cytoplasmic NO activated guanylate cyclase
• NO is a short lived gas -> very local cell to cell signaling molecule
• NO synthase is under control of Calcium calmodulin activated NO synthase