Limited proteolysis and differential down-regulation of protein kinase C subspecies in KM3 cells.

Abstract

Protein kinase C (PKC) is known to play crucial roles in signal transduction. This enzyme was originally found as an undefined protein kinase, which could be activated by limited proteolysis by a Ca2(+)-dependent neutral protease, calpain. PKC was thought to be a single entity for many years, but recent, molecular cloning and biochemical analysis has shown that this enzyme consists of multiple subspecies with closely related structures. The present studies were undertaken to explore the susceptibility of PKC subspecies to proteolysis by calpain in KM3 cells, a pre-B, pre-T cell line. The PKC from KM3 cells was resolved into two subspecies, type II (mainly beta II) and type III (alpha), upon hydroxyapatite column chromatography. This cell line was also shown to contain calpain I and calpain II, which show different sensitivities to Ca2+. Both type II and type III PKC were hydrolysed by either calpain I or calpain II, but type II PKC was cleaved more effectively than type III. The simultaneous presence of phospholipid and diacylglycerol enhanced the cleavage of these PKC subspecies by calpain I. When these cells were treated with phorbol ester, the rates of translocation and down-regulation of these PKC subspecies were different; type II PKC was translocated and depleted from the cell more rapidly than type III enzyme. The results presented herein suggest different roles of the PKC subspecies in the cellular responses to external stimuli.