A critical intramolecular interaction for protein kinase Cepsilon translocation.

Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive epsilonPKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed psiepsilonRACK. An amino acid difference between the psiepsilonRACK sequence in epsilonPKC and its homologous sequence in epsilonRACK constitutes a change from a polar non-charged amino acid (asparagine) in epsilonRACK to a polar charged amino acid (aspartate) in epsilonPKC. Here we show that mutating the aspartate to asparagine in epsilonPKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone- or PMA-induced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to epsilonRACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the psiepsilonRACK site and RACK-binding site within epsilonPKC is critical and rate limiting in the process of PKC translocation.