Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells.
Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-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 Cmu (PKCmu) 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 PKCmu 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 PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. 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.
Häussermann
S
S
German Cancer Research Center, Heidelberg.
Kittstein
W
W
Rincke
G
G
Johannes
F J
FJ
Marks
F
F
Gschwendt
M
M
D017209:Apoptosis; D054713:Bryostatins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002474:Cell-Free System; D003513:Cycloheximide / Q000494:pharmacology; D003561:Cytarabine / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D006801:Humans; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D009842:Oligopeptides / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020298:U937 Cells
D054713:Bryostatins; D007783:Lactones; D018942:Macrolides; D009842:Oligopeptides; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D003561:Cytarabine; C046785:bryostatin 1; D003513:Cycloheximide; C089212:protein kinase D; D011493:Protein Kinase C; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D005634:Fructose-Bisphosphate Aldolase