Proteolytic cleavage of protein kinase Cμ upon induction of apoptosis in U937 cells -: Identification of the cleavage site and characterization of the fragment

Treatment of U937 cells with various apoptosis-inducing agents, such as TNF alpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin I or cycloheximide, causes proteolytic cleavage of protein kinase C mu (PKC mu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase, The formation of this fragment is effectively suppressed by the caspase-3 inhibitor ZDEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKC mu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62), Treatment of several aspartic acid to alanine mutants of PKC mu with caspase-3 resulted in an unexpected finding. PKC mu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND(378)/S-379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified, In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively, (C) 1999 Federation of European Biochemical Societies.