Bryostatin 1 and phorbol ester down-modulate protein kinase C-alpha and -epsilon via the ubiquitin/proteasome pathway in human fibroblasts

We evaluated the possibility that distinct proteolytic pathways contribute to the down-regulation of a novel (epsilon) or conventional (alpha) isoform of protein kinase C (PKC) in nonimmortalized human fibroblasts. Inhibitors of calpains and other cysteine proteinases, vesicle trafficking, or lysoso...

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Bibliographic Details
Published in:Molecular pharmacology 1997-03, Vol.51 (3), p.439
Main Authors: Lee, H W, Smith, L, Pettit, G R, Smith, J B
Format: Article
Language:English
Subjects:
Acetylcysteine - analogs & derivatives
Acetylcysteine - pharmacology
Bryostatins
Cells, Cultured
Cysteine Proteinase Inhibitors - pharmacology
Down-Regulation
Fibroblasts - enzymology
Glycoproteins - pharmacology
Humans
Isoenzymes - metabolism
Lactones - pharmacology
Macrolides
Protein Kinase C - metabolism
Protein Kinase C-alpha
Protein Kinase C-epsilon
Tetradecanoylphorbol Acetate - pharmacology
Ubiquitins - metabolism
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Summary:We evaluated the possibility that distinct proteolytic pathways contribute to the down-regulation of a novel (epsilon) or conventional (alpha) isoform of protein kinase C (PKC) in nonimmortalized human fibroblasts. Inhibitors of calpains and other cysteine proteinases, vesicle trafficking, or lysosomal proteolysis did not affect the down-regulation of PKC-alpha or -epsilon produced by bryostatin 1 (Bryo). Lactacystin (Lacta) and certain terminal aldehyde tripeptides or tetrapeptides, which selectively inhibit the proteasome, preserved substantial PKC-alpha and -epsilon protein from down-regulation by Bryo or phorbol-12-myristate-13-acetate. Lacta preserved active kinase in vivo, as shown by the retention of Bryo-induced autophosphorylated PKC-alpha. Concomitant with down-regulation, Bryo produced PKC-alpha and -epsilon species that were larger than the native proteins (80 and 90 kDa, respectively). Western blot analysis showed that the larger PKC-alpha species were ubiquitinylated. Treatment with Bryo plus Lacta synergistically increased multiubiquitinylated PKC-alpha, as expected if Bryo induces ubiquitinylation of PKC-alpha and Lacta blocks its degradation. Bryo also produced a 76-kDa, nonphosphorylated form of PKC-alpha and an 86-kDa form of PKC-epsilon. Phosphatase inhibitors decreased production of 76- and 86-kDa PKC-alpha and -epsilon by Bryo and preserved 80- and 90-kDa PKC-alpha and -epsilon, respectively. Our results suggest that the down-modulation of PKC-alpha and -epsilon occurs principally via the ubiquitin/ proteasome pathway. Dephosphorylation seems to predispose PKC to ubiquitinylation.
ISSN:0026-895X