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.

Expression of an ATP binding mutant of PKC-delta inhibits Sis-induced transformation of NIH3T3 cells.

MICHIELI, Paolo;
1996-01-01

Abstract

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.
1996
13
731
737
Li W;Michieli P;Alimandi M;Lorenzi MV;Wu Y;Wang LH;Heidaran MA;Pierce JH
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/149420
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