The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation.

Dominant negative properties are conferred on protein kinase (PK) Calpha by mutation of the phosphorylation site in the activation loop of the kinase domain. To address the universality and/or specificity of such mutations, analogous alterations were introduced in other members of the PKC family and tested for their effects on the function of co-transfected activated PKC. For all three subclasses of the PKC family, mutations of the predicted activation loop phosphorylation sites resulted in dominant negative properties. These properties were not restricted to the cognate PKC isotypes, but were effective across the different subclasses. For example, two PKCzeta mutants (atypical isotype) inhibited both PKCalpha (classical isotype) and PKCepsilon (novel isotype). For all these mutants, inhibition correlated with an ability to prevent the accumulation of phosphorylated PKCalpha, consistent with the expected mode of action. In the case of the PKCalpha mutant, it was shown that inhibition required the full-length mutant protein. The results provide evidence for the involvement of a common step in the phosphorylation of all PKC isotypes.