THE BROAD-SPECIFICITY OF DOMINANT INHIBITORY PROTEIN-KINASE-C MUTANTSINFERS A COMMON STEP IN PHOSPHORYLATION

Dominant negative properties are conferred on protein kinase (PK) C al pha by mutation of the phosphorylation site in the activation loop of the kinase domain. To address the universality and/or specificity of s uch mutations, analogous alterations were introduced in other members of the PKC family and tested for their effects on the function of co-t ransfected activated PKC. For all three subclasses of the PKC family, mutations of the predicted activation loop phosphorylation sites resul ted in dominant negative properties. These properties were not restric ted to the cognate PKC isotypes, but were effective across the differe nt subclasses. For example, two PKC zeta mutants (atypical isotype) in hibited both PKC alpha (classical isotype) and PKC epsilon (novel isot ype). For all these mutants, inhibition correlated with an ability to prevent the accumulation of phosphorylated PKC alpha, consistent with the expected mode of action. In the case of the PKC alpha mutant, it w as shown that inhibition required the full-length mutant protein. The results provide evidence for the involvement of a common step in the p hosphorylation of all PKC isotypes.