A Critical Intramolecular Interaction for Protein Kinase Cε Translocation

Abstract

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 εPKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed ψεRACK. An amino acid difference between the ψεRACK sequence in εPKC and its homologous sequence in εRACK constitutes a change from a polar non-charged amino acid (asparagine) in εRACK to a polar charged amino acid (aspartate) in εPKC. Here we show that mutating the aspartate to asparagine in εPKC 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 εRACK 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 ψεRACK site and RACK-binding site within εPKC is critical and rate limiting in the process of PKC translocation.