Protein kinase C mu is regulated by the multifunctional chaperon protein p32

We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKC alpha, PKC zeta, PKC delta, an d PKC mu. We have analyzed the interaction of PKC mu with p32 in detail, an d we show here in vivo association of PKC mu, as revealed from yeast two-hy brid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC mu is constitutively associated with p32 at mitochondrial membranes, evident fro m colocalization with cytochrome c, p32 interacts with PKC mu in a compartm ent-specific manner, as it can be coimmunoprecipitated mainly from the part iculate and not from the soluble fraction, despite the presence of p32 in b oth fractions. Although p32 binds to the kinase domain of PKC mu, it does n ot serve as a substrate. Interestingly, PKC mu-p32 immunocomplexes precipit ated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation, In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays block ed, in a dose-dependent manner, aldolase but not autophosphorylation of PKC mu, suggesting a steric hindrance of substrate within the kinase domain. T ogether, these findings identify p32 as a novel, compartment-specific regul ator of PKC mu kinase activity.