One PKC target that controls cortical actin structure

One PKC target that controls cortical secretin receptor structure is a well-known actin crosslinking protein, myristoylated alanine-rich C-kinase substrate (MARCKS). MARCKS cross-links actin and binds PIP2 and this binding activity is regulated by PKC phosphorylation [82]. Activation of PKC releases MARCKS from the plasma membrane and has been directly correlated with cortical actin disassembly in chromaffin cells [47], [76]. In insulin-secreting cells, the translocation of PKC to the plasma membrane and release of MARCKS has been directly visualized [46]. These studies demonstrate that a global effect of PKC activation is the re-organization of the cortical actin cytoskeleton through loss of MARCKS from the plasma membrane. In addition to MARCKS, several other proteins directly modified by PKC are likely playing a role in actin rearrangement and vesicle movement. Vesicle-localized myosin may be regulated via PKC, perhaps via myosin light chain kinase (MLCK), which is a PKC target [83]. 14-3-3 protein was originally identified as playing a PKC-dependent role in exocytosis as well [42], [84]. Subsequent work has shown 14-3-3 likely acts as a scaffolding factor between vesicle-bound Rab-effector proteins and other molecules, such as myosin, however, it may be dependent on other kinase systems [85], [86]. Another possible target of PKC is the plasma membrane calcium channels necessary for insulin release. Modulation of these channels could provide a direct mechanism to increase the calcium response to glucose metabolism and consequently increase the exocytotic response. Beta cells have L-type, R-type, and P/Q-type calcium channels [87]. L-type channels are the primary drivers, though are not required, for GSIS [87], [88], [89], [90]. Early reports indicated PKC activation could cause calcium influx and inhibition of PKC could block exocytosis [33], [91], [92], [93], however, these effects may have been caused by intracellular calcium stores [93], [94]. The current weight of evidence shows no change of intracellular calcium levels, or indeed inhibition of calcium currents, upon PKC activation leading to the conclusion that PKC does not directly modulate calcium levels in the cell [34], [51], [52], [53], [59], [95]. Additional confusion likely also arose due to phorbol ester PKC-independent effects on channel behavior [96], [97]. PKC’s primary effect is to enhance the calcium sensitivity of exocytosis rather than to amplify or modulate calcium currents via channel activity. To the extent that PKC may influence calcium channels, it could regulate interactions between calcium channels and binding partners, which could affect exocytosis without causing large changes in calcium currents. Yokohama et al. demonstrated that Cav2.2 channels are direct PKC targets, and that phosphorylation modulates interaction of syntaxin with the channel [98]. Indeed, deletion of the syntaxin interaction site on these channels or expression of a peptide competitor for the interaction site blocks exocytosis, pointing to an important role for PKC’s modulation of this interaction [99], [100]. If syntaxin interaction with Cav2.2 primarily serves to localize the channel to the exocytotic machinery [101], rather than modulating channel activity, this may reconcile the findings that PKC does not affect calcium channel currents but that channel modification by PKC is an important regulatory mechanism. Proximity to calcium channels would provide syntaxin and the exocytotic machinery with high local calcium concentration, which is hypothesized to be a mechanism for generating the immediately-releasable pool of exocytotic vesicles [102]. Importantly, it was recently found that this pool of vesicles is absent in cells from human donors with type II diabetes [67], which strongly suggests that modulation of the L-type channel-syntaxin interaction by PKC could be critically important in diabetes. Finally, PKC can readily affect cell metabolism, which has an indirect impact on insulin secretion [103]. Several studies have shown some PKC isoforms target transcription factors [24], [104], [105]. These factors regulate the expression of metabolic factors, such as hexokinase, and consequently influence calcium currents and insulin release along the standard GSIS pathway by affecting ATP levels in the cell [22]. For example, generation of a PKC-ε knockout-mouse showed that this specific PKC likely plays a role in regulating metabolic pathways in beta cells [66]. Insulin protein synthesis is not necessary for continued insulin secretion from islets for several hours [2], [4], so it is unlikely that any PKC effects on protein synthesis would be relevant to insulin release over this timescale. Overall, the global effects of PKC regulation on cell metabolism must be very complex and more work is needed to understand PKC’s contribution to cellular ATP levels.