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Proteolytic cleavage of protein kinase Cμ upon induction of apoptosis in U937 cells
Author(s) -
Häussermann Sabine,
Kittstein Walter,
Rincke Gabriele,
Johannes Franz-Josef,
Marks Friedrich,
Gschwendt Michael
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01577-x
Subject(s) - cycloheximide , protein kinase a , protein kinase c , cleavage (geology) , phosphatidylserine , biochemistry , microbiology and biotechnology , kinase , caspase , phosphorylation , staurosporine , bryostatin 1 , u937 cell , biology , chemistry , apoptosis , protein biosynthesis , programmed cell death , fracture (geology) , paleontology , phospholipid , membrane
Treatment of U937 cells with various apoptosis‐inducing agents, such as TNFα and β‐ D ‐arabinofuranosylcytosine (ara‐C) alone or in combination with the phorbol ester 12‐ O ‐tetradecanoylphorbol‐13‐acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cμ (PKCμ) 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 Z‐DEVD‐FMK. In accordance with these in vivo data, treatment of recombinant PKCμ 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 PKCμ with caspase‐3 resulted in an unexpected finding. PKCμ 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.