Biosynthesis and posttranslational modifications of protein kinase C in human breast cancer cells.
Several forms of protein kinase C with molecular masses of 74-, 77-, and 80-kDa were detected in subcellular fractions of human breast cancer MDA-MB-231 cells which express the alpha-type protein kinase C. Several lines of evidence indicated that the 74-kDa is the precursor of the 77- and 80-kDa protein kinase C forms. (i) Pulse-labeling experiments revealed that protein kinase C is synthesized on membranes as a 74-kDa protein that can be chased into the 77- and the 80-kDa protein kinase C forms. (ii) The primary translation product of protein kinase C displayed an apparent molecular size of 74-kDa as determined by in vitro translation of poly(A)+ RNA from MDA-MB-231 cells. (iii) Incubation with serine/threonine-specific protein phosphatases (potato acid phosphatase and phosphatase 1 or 2A) resulted in the complete dephosphorylation of the 77-kDa to the 74-kDa protein kinase C form. Protein kinase C appears to be synthesized in membranes as an unphosphorylated and presumably inactive 74-kDa form that is converted into the active 77- and 80-kDa protein kinase C by post-translational modification involving at least two phosphorylation steps. The first phosphorylation is probably achieved by a specific, yet unidentified, "protein kinase C kinase" since the 74-kDa protein kinase C species did not undergo autophosphorylation and was neither a substrate for the purified protein kinase C, S6 kinase, phosphorylase kinase, casein kinase II, nor for the catalytic subunit of cAMP-dependent protein kinase. Except for phosphorylase kinase and the catalytic subunit of the cAMP-dependent protein kinase, phosphorylation of the 77-kDa protein kinase C form with purified protein kinase C (autophosphorylation), S6 kinase or casein kinase II shifted the molecular mass of the 77-kDa protein kinase C to 80-kDa. Prolonged exposure of MDA-MB-231 cells to phorbol 12-myristate 13-acetate not only leads to a complete down-regulation of protein kinase C activity but also to an accumulation of 74-kDa protein kinase C due to a retarded conversion of the 74-kDa into the 77- and 80-kDa protein kinase C forms in these cells. Our data indicate that tumor promoters additionally interfere with the posttranslational processing that converts the 74-kDa protein kinase C precursor into the 77- and 80-kDa forms of the enzyme.
Borner
C
C
Department of Research, University Medical School, Basel, Switzerland.
Filipuzzi
I
I
Wartmann
M
M
Eppenberger
U
U
Fabbro
D
D
D001943:Breast Neoplasms / Q000201:enzymology*; D002460:Cell Line; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008962:Models, Theoretical; D008970:Molecular Weight; D010766:Phosphorylation; D011061:Poly A / Q000235:genetics; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D014407:Tumor Cells, Cultured / Q000201:enzymology*
D007527:Isoenzymes; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D011494:Protein Kinases; D011493:Protein Kinase C