Phosphorylation of protein kinase C-zeta on serine 657 controls the accumulation of active enzyme and contributes to its phosphatase-resistant state

Serine 657 in protein kinase C-alpha (PKC alpha) is a site of phosphorylation on expression of the recombinant protein in mammalian cells. To define the function of this phosphorylation, PKC alpha species with mutations of this site were investigated. The alanine mutant, S657A PKC alpha, displayed slow phosphate accumulation in pulse-chase experiments, indicating a rate-limiting role in the initial phase of phosphorylation. Consistent with this, the aspartic acid mutant, S657D PKC alpha, showed an increased rate of phosphate accumulation. Both the S657D and S657A PKC alpha mutants were slow to accumulate as fully phosphorylated forms during a second phase of phosphorylation. This latter property is shown to correlate with an increased phosphatase sensitivity and decreased protein kinase activity for these two PKC alpha mutants. It is further shown that once fully phosphorylated, the S657D PKC alpha mutant displays WT PKC alpha properties with respect to thermal stability and phosphatase sensitivity in vitro and in vivo; in contrast, the S657A PKC alpha mutant remains sensitive. The properties of the Ser-657 site PKC alpha mutants define functional roles for this phosphorylation in both the accumulation of phosphate on PKC alpha as well as in its agonist-induced dephosphorylation. These results are discussed in the context of a working model of PKC alpha behavior, providing insight into the workings of other kinases with equivalent sites of phosphorylation.