NOVEL-TYPE PROTEIN KINASE C ISOFORM-SPECIFIC FUNCTION, KINASE ACTIVITY, AND PHOSPHORYLATION STATUS ARE DISRUPTED BY V5 DOMAIN MUTATIONS
Schlapkohl, Walter Antonio
Mushinski, J. Frederic
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The Protein Kinase C (PKC) family consists of at least 10 different isoforms that are subdivided into three groups: "classical/conventional" cPKCs (alpha, betaI-II and gamma), "novel" nPKCs (delta, epsilon, eta and theta) and "atypical" aPKCs (zeta and lambda/iota). The nPKCs are "novel" because they do not respond to changes in calcium levels as do the classical PKCs, but respond only to changes in diacylglycerol levels. This work elucidates several aspects of the V5 domain's role in nPKC function through the use of PKC-delta and -epsilon truncation mutants, as well as two reciprocal PKC-delta and -epsilon C-terminal chimeras: PKC-delta/epsilonV5 and PKC-epsilon/deltaV5. First, the ability of the V5 domain to confer isoform-specific function was tested by overexpressing chimeric PKC mutants in 32D cells. Overexpression of wild-type PKC-delta, but not PKC-epsilon, mediated macrophage differentiation in this cell line upon stimulation with TPA. However, neither PKC-detal/epsilonV5 nor -epsilon/deltaV5 were able to confer a TPA-inducible macrophage differentiation phenotype to 32D cells, indicating that PKC-delta V5 domain was essential to this isoform-specific function, but not sufficient to transfer its activity to PKC-epsilon. Second, nPKC mutants were utilized to determine the ability of the V5 domain to modulate kinase activity. PKC chimeric and truncation mutants had significantly decreased kinase activity in vitro compared to their wild-type counterparts. PKC-delta/epsilonV5 and -epsilon/deltaV5 lacked phosphorylation at critical phosphorylation priming sites, a likely cause of their reduced kinase activity. Glutamic acid substitutions made at unphosphorylated priming sites, either individually or in combination, did not rescue kinase activity. Stimulation with TPA increased the level of phosphorylation only at sites that were already phosphorylated. The lack of phosphorylation and kinase activity are likely due to differences in phosphatase activity, since both PKC-delta and -epsilon V5 chimeras bound the upstream, activating kinase, Phosphoinositide-dependent Kinase-1. In conclusion, changes to the V5 domain of PKC-delta and -epsilon disrupted the proteins' phosphorylation status and kinase function which, in turn, likely prevented conferral of isoform-specific function in a macrophage differentiation assay with PKC-delta and -epsilon V5 chimeras.