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 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.
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
Smith
J B
JB
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D054713:Bryostatins; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation; D005347:Fibroblasts / Q000201:enzymology; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology*; D018942:Macrolides; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014452:Ubiquitins / Q000378:metabolism
D054713:Bryostatins; D015853:Cysteine Proteinase Inhibitors; D006023:Glycoproteins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D014452:Ubiquitins; C039304:calpain inhibitors; C067713:lactacystin; C046785:bryostatin 1; C496628:PRKCA protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate; D000111:Acetylcysteine