Activation Cycle of Protein Kinase C
Conventional isoforms of PKC are activated by the second messengers calcium and diacylglycerol (DAG). In the absence of these second messengers, PKC has a low membrane affinity and localizes primarily to the cytosol. Following agonist stimulation, calcium binds to the C2 domain (yellow), targeting it to anionic phospholipid membranes. PKC then undergoes an efficient 2-dimensional search for DAG in the plane of the membrane. Membrane-partitioned DAG and phosphatidylserine (PS) help anchor PKC to membranes by binding the C1 domain (orange). When both domains are engaged, an autoinhibitory pseudosubstrate sequence (purple) is expelled from the active site of the catalytic domain (blue). The exposed active site catalyzes the transfer of phosphate to suitably localized protein substrates. As cells return to the resting state, levels of calcium and DAG are reduced, inactivating PKC by lowering its membrane affinity.
Visualizing Akt/PKB activity
Akt/PKB activity is visualized in live cells by monitoring BKAR, a FRET-based kinase activity reporter for Akt/PKB. In this movie, following the addition of EGF one can observe the activation of Akt/PKB through a FRET change from BKAR. That is, BKAR is unphosphorylated prior to EGF treatment (blue/green) and becomes phosphorylated following EGF addition and subsequent Akt/PKB activation (yellow/red). The BKAR FRET ratio returns to baseline (dephosphorylated, blue/green) following inhibition of the Akt/PKB pathway.