Protein kinase C isozymes in progressively transformed rat embryo fibroblasts.
The role of individual protein kinase C (PKC) isoforms in progressive transformation of a rat embryo fibroblast cell line (REF52 cells) has been evaluated. Normal (REF A) cells were transfected with SV40 to produce the partially transformed REF B cell line. REF B cells are morphologically transformed but have only limited capacity for growth in soft agar. Message and protein levels for PKC-alpha and PKC-delta were similar in REF A and B cells, indicating that expression of SV40 large T did not directly influence the amounts of PKCs. However, PKC-alpha localization was influenced. PKC-alpha was associated with focal contacts of REF A but not REF B cells, indicating that changes in location rather than content are an early event in REF52 cell transformation. Clones of REF B cells were selected for growth in soft agar (REF C cells). Levels of PKC-delta, but not PKC-alpha or epsilon, were increased in several of these clones, suggesting that increased PKC-delta content may facilitate anchorage-independent growth. In other studies, we have determined that PKCs interact with their binding proteins/substrates through their regulatory domains (RD; L. Liao et al., Biochemistry, 33: 1229-1233, 1994). These results predict that RDs could potentially inhibit PKC signaling by competing with endogenous wild-type PKCs for binding protein/substrate interactions. Overexpression of the RD of PKC-delta inhibited growth in soft agar of one representative REF C clone, whereas overexpression of the RD of PKC-alpha promoted growth in soft agar. These results suggest that RD expression may be a useful approach for dominant negative PKC inhibitors with potential isozyme specificity.
Liao
L
L
W. Alton Jones Cell Science Center, Inc., Lake Placid, New York 12946.
Ramsay
K
K
Jaken
S
S
D000818:Animals; D002455:Cell Division; D002458:Cell Fractionation; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000821:virology; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051381:Rats; D013539:Simian virus 40 / Q000502:physiology; D014162:Transfection
D007527:Isoenzymes; D015240:Phorbol 12,13-Dibutyrate; C497260:Prkcd protein, rat; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta