Degradation of protein kinase C alpha and its free catalytic subunit, protein kinase M, in intact human neuroblastoma cells and under cell-free conditions. Evidence that PKM is degraded by mM calpain-mediated proteolysis at a faster rate than PKC.
Proteolytic cleavage of protein kinase C (PKC) under cell-free conditions generates a co-factor independent, free catalytic subunit (PKM). However, the difficulty in visualizing PKM in intact cells has generated controversy regarding its physiological relevance. In the present study, treatment of SH-SY-5Y cells with 2-O-tetradecanoylphorbol 13-acetate resulted in complete down-regulation of PKC within 24 h without detection of PKM. By contrast, low levels of PKM were transiently detected following ionophore-mediated calcium influx under conditions which induced no detectable PKC loss. PKM was not detected during rapid cell-free degradation of partially purified SH-SY-5Y PKC alpha by purified human brain mM calpain. However, when the kinetics of PKC degradation were slowed by lowering levels of calpain, PKM was transiently detected. PKM was also only transiently observed following calpain-mediated degradation of purified rat brain PKC alpha. Densitometric analyses indicated that, once formed, PKM was degraded approximately 10 times faster than PKC. These data provide an explanation as to why PKM is difficult to observe in situ, and indicate that PKM should not be considered as an 'unregulated' kinase, since its persistence is apparently strictly regulated by proteolysis.
Shea
T B
TB
Laboratories for Molecular Neuroscience, Mailman Research Center, McLean Hospital, Belmont, MA 02178.
Beermann
M L
ML
Griffin
W R
WR
Leli
U
U
D002154:Calpain / Q000378:metabolism*; D002474:Cell-Free System; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D009447:Neuroblastoma / Q000201:enzymology; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002154:Calpain; D013755:Tetradecanoylphorbol Acetate