Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin.
We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-alpha (PKC-alpha) in the LLC-MK2 line of renal epithelial cells. Bryo, like phorbol 12-myristate 13-acetate (PMA), acutely activated PKC, as indicated by autophosphorylation and translocation of PKC-alpha, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-alpha by autophosphorylation and produced a 76-kDa form of PKC-alpha that lacked detectable 32P. The 76-kDa form was in the particulate rather than the cytosolic fraction, which suggests that it was produced from activated kinase. Alkaline phosphatase treatment of immunoprecipitated PKC-alpha converted the 80-kDa form to 76 kDa, but it had no effect on the mobility of the 76-kDa form, suggesting that it was not phosphorylated. Pulse-chase labeling of PKC-alpha with [35S]Met/Cys indicated that there is a precursor-product relationship between the 80- and 76-kDa forms, respectively. Inhibition of protein synthesis had no effect on the production of 76-kDa PKC-alpha by Bryo. PMA also produced 76-kDa PKC-alpha but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-alpha protein and total Ca(2+)- and phospholipid-dependent PKC activity than PMA. The 76-kDa form is inactive and incompetent because it lacked detectable 32P under conditions that strongly autophosphorylated the 80-kDa form. We suggest that dephosphorylation predisposes PKC to proteolysis, and greater production of the 76-kDa form explains the more efficient downregulation of the kinase by Bryo vs. PMA.
Lee
H W
HW
Department of Pharmacology and Toxicology, School of Medicine, University of Alabama at Birmingham 35294, USA.
Smith
L
L
Pettit
G R
GR
Bingham Smith
J
J
D000469:Alkaline Phosphatase / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D054713:Bryostatins; D002460:Cell Line; D004789:Enzyme Activation; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007783:Lactones / Q000494:pharmacology*; D018942:Macrolides; D008970:Molecular Weight; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000970:Antineoplastic Agents; D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; C046785:bryostatin 1; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D013755:Tetradecanoylphorbol Acetate