Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin

Hyeon Woo Lee, Lucinda Smith, George Pettit, Jeffrey Bingham Smith

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-α (PKC-α) 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-α, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-α by autophosphorylation and produced a 76-kDa form of PKC-a 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-a 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-a 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-a by Bryo. PMA also produced 76-kDa PKC-α but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-α protein and total Ca2+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.

Original languageEnglish (US)
Pages (from-to)C312-C320
JournalAmerican Journal of Physiology
Volume271
Issue number1 PART 1
StatePublished - Dec 1 1996

Keywords

  • Kidney
  • Phorbol 12-myristate 13-acetate
  • Tumor promoter

ASJC Scopus subject areas

  • Physiology (medical)

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