Cells sense the changes in their environment through signal cascades initiated by receptors on the outer cell membrane. The signal transduction pathway that activates PKC consists of receptor-mediated activation of phopsholipases C, leading to hydrolysis of PtdIns4,5
to produce DAG, as well as a rise in intracellular calcium levels (12
). Inactive PKCs are found in the soluble fraction of the cells, and translocate upon increases in cellular signaling to various membrane surfaces, where they bind anchoring molecules and phosphorylate neighboring protein substrates (5
The current model for the activation of the classical PKC isozymes suggests that calcium binding increases the affinity of PKC for phosphatidylserine (PS) at the cell membrane. This, in turn, enables the kinase to laterally ‘search’ for and bind DAG molecules that are found at low abundance in the membrane (13
). The binding of the two regulatory domains to the membrane releases the auto-inhibitory pseudosubstrate site (Ψsubstrate) from the active site in the catalytic domain, producing conformational changes that leave the catalytic domain accessible to substrate binding and phosphorylation. The novel family of PKCs do not bind calcium, but have higher affinity for DAG as compared to the classical PKC family (12
), producing a fine balance of responsiveness to similar activators for different isozymes. In addition to second-messenger sensing domains, other PKC regions and domains, described below, participate in PKC activation and subcellular localization, resulting in multi-step events leading to PKC activation, localization and function.
Multiple PKC isozymes can be present in the same cell, and can translocate to different subcellular localizations in response to the same stimuli (6
). In order to explain this phenomenon, it was hypothesized that each individual PKC isozyme might have an isozyme-selective anchoring protein to which each PKC isozyme binds upon activation. These anchoring proteins, termed r
eceptors for a
ctivated C K
inase (RACKs), are hypothesized to anchor specific PKC isozymes at unique subcellular locations (5
). Thus, anchoring of a specific PKC isozyme to its respective RACK localizes that PKC isozyme in close proximity to its isozyme-specific protein substrates. Subcellular translocation and binding to isozyme-selective RACKs can therefore bestow functional specificity for each PKC isozyme. Two RACKs have been identified to date: the RACK for βIIPKC, known as RACK1 (14
), and the RACK for εPKC, known as RACK2 or β′COP (15
). The specificity of PKC-RACK interaction is thought to be mediated by the C2 and the V5 domains, discussed in detail below, though further characterization of these proteins may elucidate more isozyme-selective interaction sites.
In order to better understand isozyme-specific roles and activation mechanisms, whole enzyme and individual PKC domains are used to study protein-protein interactions and translocation of PKC isozymes. The complexity of PKC signaling becomes increasingly apparent as our understanding of the mechanism of PKC function is elucidated. For example, the mechanism underlying PKC movement from the cytosol to the membrane is still debated. There is evidence that PKC translocation is dependent on cytoskeletal elements (16
), yet studies calculating the accumulation of PKC at the membrane suggest that the translocation process is diffusion-limited (e.g., (18
)). However, studies concluding that PKC translocation depends only on the speed of diffusion utilized over-expressed tagged PKC, whereas those favoring active transport were conducted with endogenous proteins. It is therefore possible that over-expression saturates the putative machinery required for active PKC translocation. If PKC translocation involves active transport along the cytoskeletal elements, a new set of protein-protein interactions may be involved in the translocation mechanism.