Protein kinase C (PKC) is a key regulator of cell proliferation, differentiation, and apoptosis and is one of the drug targets of anticancer therapy. Recently, a single point mutation (D294G) in PKCA has been found in pituitary and thyroid tumors with more invasive phenotype. Although the PKCA-D294G mutant is implicated in the progression of endocrine tumors, no apparent biochemical/cell biological abnormalities underlying tumorigenesis with this mutant have been found. We report here that the PKCA-D294G mutant is unable to bind to cellular membranes tightly despite the fact that it translocates to the membrane as efficiently as the wild-type PKCA upon treatment of phorbol ester. The impaired membrane binding is associated with this mutant's inability to transduce several antitumorigenic signals as it fails to mediate phorbol ester-stimulated translocation of myristoylated alanine-rich protein kinase C substrate (MARCKS), to activate mitogen-activated protein kinase and to augment melatonin-stimulated neurite outgrowth. Thus, the PKCA-D294G is a loss-of-function mutation. We propose that the wild-type PKCA may play important antitumorigenic roles in the progression of endocrine tumors. Therefore, developing selective activators instead of inhibitors of PKCA might provide effective pharmacological interventions for the treatment of certain endocrine tumors. (Cancer Res 2005; 65(11): 4520-4)

The segment C-terminal to the hydrophobic motif at the V5 domain of protein kinase C (PKC) is the least conserved both in length and in amino acid identity among all PKC isozymes. By generating serial truncation mutants followed by biochemical and functional analyses, we show here that the very C terminus of PKC␣ is critical in conferring the full catalytic competence to the kinase and for transducing signals in cells. Deletion of one C-terminal amino acid residue caused the loss of ϳ60% of the catalytic activity of the mutant PKC␣, whereas deletion of 10 C-terminal amino acid residues abrogated the catalytic activity of PKC␣ in immune complex kinase assays. The PKC␣ C-terminal truncation mutants were found to lose their ability to activate mitogen-activated protein kinase, to rescue apoptosis induced by the inhibition of endogenous PKC in COS cells, and to augment melatonin-stimulated neurite outgrowth. Furthermore, molecular dynamics simulations revealed that the deletion of 1 or 10 C-terminal residues results in the deformation of the V5 domain and the ATP-binding pocket, respectively. Finally, PKC␣ immunoprecipitated using an antibody against its C terminus had only marginal catalytic activity compared with that of the PKC␣ immunoprecipitated by an antibody against its N terminus. Therefore, the very C-terminal tail of PKC␣ is a novel determinant of the catalytic activity of PKC and a promising target for selective modulation of PKC␣ function. Molecules that bind preferentially to the very C terminus of distinct PKC isozymes and suppress their catalytic activity may constitute a new class of selective inhibitors of PKC.

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