Fuller S J, Finn S G, Downward J, Sugden P H
NHLI Division (Cardiac Medicine), Imperial College School of Medicine, London SW3 6LY, UK.
Biochem J. 1998 Oct 15;335 ( Pt 2)(Pt 2):241-6. doi: 10.1042/bj3350241.
Treatment of cultured neonatal ventricular myocytes with oncogenic Ras increases their size and stimulates the re-expression of genes which are normally restricted to the fetal stage of ventricular development, including atrial natriuretic factor (ANF) and skeletal muscle (SkM)-alpha-actin. To determine which signalling pathways mediate these responses, myocytes were transfected with oncogenic (V12) Ras mutants which interact selectively with different effectors and their effects on luciferase (LUX) reporter plasmids were examined. V12 human Ras (V12HRas), itself, activated ANF-LUX 9. 6-fold, whereas mutants of V12HRas, which selectively stimulate Ral guanine nucleotide dissociation stimulator (Ral.GDS) (E37G), c-Raf (D38E) and phosphatidylinositol 3-kinase (PI-3-K; Y40C) enhanced ANF-LUX expression 3.0-, 3.7- and 1.7-fold respectively. The full response of ANF-LUX to V12HRas was restored by using a combination of the individual effector domain mutants. Likewise, SkM-alpha-actin-LUX expression was activated 12.0-, 3.5-, 4.5- and 3. 0-fold by V12HRas, E37G, D38E and Y40C respectively, and a similar pattern of activation was also observed using a c-fos serum-response element-LUX reporter gene. Cell size was also increased by each of the mutants, but simultaneous expression of all three mutant constructs was needed to reconstitute the full effect of V12HRas on cell size (50% increase). Transfection with a constitutively active mutant of PI-3-K (p110K227E) stimulated ANF-LUX, SkM-alpha-actin-LUX, c-fos-serum-response element-LUX and Rous sarcoma virus-LUX by 3.1-, 3.2-, 2.1- and 2.9-fold respectively, but the co-transfected cytomegalovirus-beta-galactosidase reporter gene was activated to a similar extent (1.9-fold). These results suggest that Raf, Ral.GDS and PI-3-K can all transduce transcriptional responses to V12HRas, but that the specific induction of genes associated with the hypertrophic response is not mediated through PI-3-K.
用致癌性Ras处理培养的新生大鼠心室肌细胞会增加其大小，并刺激通常仅限于心室发育胎儿期的基因重新表达，包括心钠素（ANF）和骨骼肌（SkM）α-肌动蛋白。为了确定哪些信号通路介导这些反应，将与不同效应器选择性相互作用的致癌性（V12）Ras突变体转染到心肌细胞中，并检测它们对荧光素酶（LUX）报告质粒的影响。V12人Ras（V12HRas）本身可激活ANF-LUX 9.6倍，而选择性刺激Ral鸟嘌呤核苷酸解离刺激剂（Ral.GDS）（E37G）、c-Raf（D38E）和磷脂酰肌醇3激酶（PI-3-K；Y40C）的V12HRas突变体分别增强ANF-LUX表达3.0倍、3.7倍和1.7倍。通过使用单个效应器结构域突变体的组合，可恢复ANF-LUX对V12HRas的完全反应。同样，V12HRas、E37G、D38E和Y40C分别激活SkM-α-肌动蛋白-LUX表达12.0倍、3.5倍、4.5倍和3.0倍，并且使用c-fos血清反应元件-LUX报告基因也观察到类似的激活模式。每个突变体也会增加细胞大小，但需要同时表达所有三种突变体构建体才能重建V12HRas对细胞大小的完全作用（增加50%）。用组成型活性PI-3-K突变体（p110K227E）转染分别刺激ANF-LUX、SkM-α-肌动蛋白-LUX、c-fos血清反应元件-LUX和劳氏肉瘤病毒-LUX 3.1倍、3.2倍、2.1倍和2.9倍，但共转染的巨细胞病毒-β-半乳糖苷酶报告基因被激活到类似程度（1.9倍）。这些结果表明，Raf、Ral.GDS和PI-3-K都可以转导对V12HRas的转录反应，但与肥厚反应相关的基因的特异性诱导不是通过PI-3-K介导的。
