8 - PKA/PKC

PKC activation

1 DAG made by PLC and Ca from stores released. 2 PKC recruited to membrane, binds DAG and Ca. 3 PKC hydrophobic domain binds PDK1 and activates it. 4 PDK1 phosphorylates to activate PKC which then autophosphorylates and is now fully active. 5 DAG kinase phosphorylates DAG, Ca drops. 6 PKC is now inactive but can be restimulated

PKC structure

C1 domain binds phorbol ester or DAG which helps anchor to membrane and has pseudosubstrate domain. C2 domain binds Ca, PO4-lipid. C3 and C4 domains are catalytic, present in all PKC families. regulatory C1/C2 domains linked by flexible hinge to C3/C4. hydrophobic motif at end regulates initial activation.

PKC families

Conventional PKC has parts to bind Ca, DAG, and phospholipids. Novel PKCs can't bind Ca. Atypical PKC only bind phospholipids. all have pseudosubstrate domain to stop inappropriate activation.

Opposing PKC action

DAG kinases modify DAG activator to shut PKC down. DAGK also have regulatory and catalytic domains. product of DAGK can recruit Raf proteins to cell membrane to activate a PKC isoform that doesn't respond to DAG. prolonged phorbol ester activation eventually induces PKC inactivation by dephosphorylation and destruction.

PKA and transcriptional regulation

PKA phosphorylates CREB target that then binds CRE elements in DNA. CRE elements are upstream from TATA box, require more protein factors than just CREB. CREB phosphorylation responsive to hormones causing Ca release. Ca also activates CaMKII which phosphorylates same site and causes release of glucagon to increase glucose.

PKC binding proteins

PKC seem to be targeted to specific areas in cells. RACK1 recruits active PKC to the ribosome where it is involved in phosphorylating elongation factors. RACK1 also interacts with membrane bound receptors to allow translocation of proteins. some membrane and cytoskeletal proteins only interact with inactive PKC. proteins that are specific to only one isoform of PKC confer specificity and can link signaling pathways.

Extracellular Regulated Kinase activation

activated B-adrenergic receptors usually transduce signals through Gs to activate AC and produce cAMP. cAMP activates PKA. one PKA target is B-adrenergic receptor. phosphorylation causes change in affinity from Gs to Gi leading to new pathway and ERK activation.

PKC introduction

activated by Ca, DAG, and phospholipids. PKC involved in many cellular processes. PKC has 3 main families each with a few isoforms and differ in the type of activator required due to structural differences. phosphorylation motif is not very selective.

Cyclic AMP and Signal Amplification

adrenaline at pM concentration increases cAMP. cAMP activates PKA, PKA activates phosphorylase kinase that activates glycogen phosphorylase. Unactivated glycogen phosphorylase can be allosterically controlled by ATP, AMP, G-6-P but modified phosphorylase can't be. PKA also phosphorylates to inactivate glycogen synthase.

2ndary PKC regulation

almost all signal molecules end up leading to production of DAG, IP3, therefore PKC is involved in many cellular processes but requires *both* DAG and sustained Ca spikes. Other lipids 2nd messengers play roles in PKC signaling. most activate, except sphinglycolipids which inhibit. different isoforms of PKC have different reactions to regulators.

Protein Kinase A

modifies multiple targets Ser/Thr with specific motif. has regulatory subunits that have pseudosubstrate inhibitory loop that can't be phosphorylated. when regulatory subunits bind cAMP, dissociate from catalytic units which are now active. affinity for cAMP is lost by reg subunits so cAMP released, destroyed. reg units again bind catalytic subunits inactivating them.