Expression of an ATP binding mutant of PKC-delta inhibits Sis-induced transformation of NIH3T3 cells.
In an effort to determine the role of protein kinase C-delta (PKC-delta) in cellular transformation mediated by the sis proto-oncogene, we cotransfected expression vectors containing cDNAs that encode for c-sis with an ATP binding mutant of PKC-delta (PKC-delta K376R) or wild type PKC-delta (PKC-delta WT) into NIH3T3 cells. Our results showed that expression of PKC-delta K376R severely impaired Sis-induced focus formation, whereas cotransfection of PKC-delta WT cDNA had no effect on Sis-mediated transformation. Consistent with this result, PKC-delta K376R expression also inhibited PDGF-BB-mediated anchorage-independent colony formation. While cotransfection of a vector containing a dominant negative mutant of ras (N17 ras) cDNA potently inhibited Sis-induced transformation, the expression of PKC-delta K376R did not block transformation mediated by v-H-Ras or v-Raf. In addition, PDGF-BB-induced Raf and mitogen-activated protein kinase activation, which are known to be downstream molecules in the Ras cascade, were not affected by the expression of PKC-delta K376R, indicating that PKC-delta and Ras are segregated in mediating Sis-induced transformation. Interestingly, expression of PKC-delta K376R strongly reduced TPA responsive element (TRE) transactivation induced by PDGF stimulation, suggesting that activation of TRE-containing genes, which may be involved in Sis-mediated transformation, are negatively regulated by expression of PKC-delta K376R.
Li
W
W
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Michieli
P
P
Alimandi
M
M
Lorenzi
M V
MV
Wu
Y
Y
Wang
L H
LH
Heidaran
M A
MA
Pierce
J H
JH
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D017931:DNA Primers; D004790:Enzyme Induction; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020574:Proto-Oncogene Proteins c-sis; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
D017931:DNA Primers; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D020574:Proto-Oncogene Proteins c-sis; D000255:Adenosine Triphosphate; D008156:Luciferases; C497259:Prkcd protein, mouse; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D018631:ras Proteins