Down-regulation of protein kinase C and of an endogenous 80-kDa substrate in transformed fibroblasts.
Subconfluent cultures of NIH-3T3 fibroblasts transformed by the Ha-ras, Ki-v-ras, v-src, and v-fms oncogene proteins all possess elevated steady-state levels of diacylglycerol, the endogenous activator of protein kinase C, as compared to the nontransformed parental lines. These oncogene-transformed fibroblasts also exhibit a significantly decreased level of cellular protein kinase C activity as measured by four different criteria: phorbol ester-stimulated phosphorylation of an endogenous 80-kilodalton (80 kDa) substrate; phorbol ester-stimulated changes in 86Rb uptake; enzymatic assay; and [3H]phorbol ester binding. In all cases, the transformed cells demonstrated an attenuated response to phorbol ester addition and a lower phorbol ester binding capacity as compared to the parental lines. Western analysis of the endogenous 80-kDa substrate of protein kinase C revealed a significantly lower level of this protein in the transformed cells than in the untransformed controls, and this decrease could be mimicked in parental cells by long-term incubation with phorbol esters, suggesting that the level of the 80-kDa protein is regulated by the state of activation of protein kinase C. These effects do not appear to be nonspecific responses to autocrine secretions by the transformed cells. They may represent an unsuccessful attempt by the transformed cells to negatively modulate the constitutive proliferative signals generated by the oncogene products.
Wolfman
A
A
Department of Biophysics, School of Medicine and Dentistry, University of Rochester, New York 14642.
Wingrove
T G
TG
Blackshear
P J
PJ
Macara
I G
IG
D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D009857:Oncogenes; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D014162:Transfection
D004075:Diglycerides; D011506:Proteins; D010042:Ouabain; D011493:Protein Kinase C; D012413:Rubidium