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

为了进一步阐明蛋白激酶C-d（PKC-d）在 PDGF介导的信号转导，一种三磷酸腺苷（ATP） 通过将恒定的赖氨酸转化成PKC-d结合突变体， 在氨基酸376处变成精氨酸。 突变体PKC-dK 376 R则没有 具有自磷酸化活性或转磷酸化能力 当在32 D细胞中表达时为外源底物。 与32 D细胞不同， 过度表达野生型PKC-d（PKC-dWT），用PKC-dWT转染的32 D细胞， dK 376 R对ATP应答不经历单核细胞分化 刺激. 此外，PKC-dK 376 R竞争性抑制PKC-dWT， 在体外PKC-d活性测定中的活性。 最后一个结果表明 ATP结合突变体可能以显性负性方式起作用， 阻断PKC-d相关的生物学功能。 载体共转染 将含有PKC-dK 376 R和原癌基因sis的重组质粒导入NIH/3 T3细胞， 严重损害sis诱导的病灶形成;而共转染 PKC-dWT或载体单独与sis一起使用对sis介导的细胞凋亡没有影响。 转型 PKC-dK 376 R/sis共转染子的肿瘤发展是 与PKC-dWT/sis诱导的那些相比，延迟10天，或 载体/SIS共转染子。 PKC-dK 376 R表达也抑制PKGF-BB 介导的锚定非依赖性集落形成。 蛋白激酶C- dK 376 R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 清楚地表明PKC-d位于PDGF-bR活化的下游， 阻断PKC-d的活化严重抑制PDGF介导的细胞凋亡。 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.