The function of PKC isozymes can be probed using activators such as tumor-promoting phorbol esters, which activate conventional and novel PKCs, although it is important to note that other proteins besides PKCs can be activated by phorbol esters.171 However, when reversed by inhibitors of PKC catalytic activity, it is possible to ascribe effects of phorbol esters to activation of PKC. The activation of PKCs can be measured as autophosphorylation of PKCs or their translocation to different cellular compartments. Several inhibitors are commonly used, such as bisindolylmaleimides and related compounds that bind to the purine site in the catalytic domain, or other compounds that do not compete with ATP binding such as chelerythrine and calphostin C. It is important to note that these inhibitors do not distinguish well between PKC isozymes, although one, the indolocarbazole Go6976, inhibits conventional PKCs with good selectivity over novel and atypical PKCs.172 Isozyme-specific manipulations have been best achieved through the use of isozyme-selective peptide inhibitors derived from putative RACK binding domains, over-expression of isozymes, expression of dominant negative mutants of specific isozymes, gene targeting or expression of PKC transgenes, antisense RNA, or RNA interference.
