Ubiquitination of protein kinase C-alpha and degradation by the proteasome.
Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.
Lee
H W
HW
Department of Pharmacology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, 35294, USA.
Smith
L
L
Pettit
G R
GR
Vinitsky
A
A
Smith
J B
JB
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D054713:Bryostatins; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D015536:Down-Regulation; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D008253:Macaca mulatta; D018942:Macrolides; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D014452:Ubiquitins / Q000378:metabolism*
D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D009097:Multienzyme Complexes; D014452:Ubiquitins; C067713:lactacystin; C046785:bryostatin 1; C035766:leucinal; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D007930:Leucine; D000111:Acetylcysteine