INHIBITION OF SIS-INDUCED TRANSFORMATION OF NIH/3T3 CELLS

为进一步阐明蛋白激酶C-d(PKC-d)在心肌梗死中的作用 PDGF介导的信号转导--三磷酸腺苷(ATP) 通过将不变的赖氨酸转化为PKC-d的结合突变体 氨基酸为376位的精氨酸。突变体PKC-dK376R没有 具有自身磷酸化活性或转磷酰化能力 在32D细胞中表达时的外源底物。与32D单元不同 高表达野生型PKC-d(PKC-DWT)，转染PKC-d的32D细胞 DK376R在ATP诱导下未发生单核细胞分化 刺激。此外，PKC-dK376R竞争性抑制PKC-DWT 体外PKC-d活性测定。这最后的结果表明 ATP结合突变体可能以显性负向方式起作用 阻断PKC-d相关的生物学功能。载体共转染 含PKC-dK376R和原癌基因sis的NIH/3T3细胞 严重损害了SIS诱导的病灶形成；而共转染 PKC-DWT或载体单独与SIS联用对SIS介导的SIS没有影响 转型。PKC-dK376R/sis共转染体的成瘤作用 与PKC-DWT/sis或PKC-DWT/sis或 载体/sis共转染体。PKC-dK376R的表达也抑制了PKGF-BB 介导不依赖锚地的菌落形成。PKC-1基因的表达 DK376R不抑制PDGF-BR自身磷酸化，但严重抑制PDGF-BR自身磷酸化 抑制PDGF诱导的早期反应基因激活。结果是 清楚地表明PKC-d位于PDGF-BR激活的下游，并且 阻断PKC-d激活严重抑制PDGF介导的细胞 转型。

To further elucidate the role played by protein kinase C-d (PKC-d) in PDGF-mediated signal transduction, an adenosine triphosphate (ATP) binding mutant of PKC-d was generated by converting the invarient lysine to an arginine at amino acid 376. The mutant, PKC-dK376R, did not possess autophosphorylation activity or the ability to transphosphorylate an exogenous substrate when expressed in 32D cells. Unlike 32D cells overexpressing wild type PKC-d (PKC-dWT), 32D cells transfected with PKC- dK376R did not undergo monocytic differentiation in response to ATP stimulation. Moreover, PKC-dK376R competitively inhibited PKC-dWT activity in an in vitro PKC-d activity assay. This last result suggests that the ATP binding mutant might act in a dominant negative manner and block PKC-d-related biological functions. Cotransfection of vectors containing PKC-dK376R and the protooncogene, sis, into NIH/3T3 cells severely impaired sis-induced focus formation; whereas cotransfection of PKC-dWT or vector alone with sis had no effect on sis-mediated transformation. Tumor development by PKC-dK376R/sis cotransfectants was delayed by 10 days when compared to those induced by PKC-dWT/sis or vector/sis cotransfectants. PKC-dK376R expression also inhibited PKGF-BB mediated anchorage-independent colony formation. Expression of PKC- dK376R did not inhibit PDGF-bR autophosphorylation, but severely inhibited PDGF-induced early response gene activation. The results clearly indicate that PKC-d lies downstream of PDGF-bR activation and blockage of PKC-d activation severely inhibits PDGF-mediated cellular transformation.