Proteolytic cleavage of protein kinase C mu upon induction of apoptosis inU937 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), bryostat in I or cycloheximide, causes proteolytic cleavage of protein kinase C mu ( PKC mu) between the regulatory and catalytic domain, generating a 62 kDa ca talytic fragment of the kinase, The formation of this fragment is effective ly suppressed by the caspase-3 inhibitor ZDEVD-FMK. In accordance with thes e in vivo data, treatment of recombinant PKC mu with caspase-3 in vitro res ults also in the generation of a 62 kDa fragment (p62), Treatment of severa l 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 si tes 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 G ST fusion protein (GST-p62) and partially purified, In contrast to the inta ct kinase, the fragment does not respond to the activating cofactors TPA an d phosphatidylserine and is thus unable to phosphorylate substrates effecti vely, (C) 1999 Federation of European Biochemical Societies.