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)-α-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-α-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-fosserum-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-α-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-β-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.
Stimulation of gene expression in neonatal rat ventricular myocytes by Ras is mediated by Ral guanine nucleotide dissociation stimulator (Ral.GDS) and phosphatidylinositol 3-kinase in addition to Raf
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Stephen J. FULLER, Stephen G. FINN, Julian DOWNWARD, Peter H. SUGDEN; Stimulation of gene expression in neonatal rat ventricular myocytes by Ras is mediated by Ral guanine nucleotide dissociation stimulator (Ral.GDS) and phosphatidylinositol 3-kinase in addition to Raf. Biochem J 15 October 1998; 335 (2): 241–246. doi: https://doi.org/10.1042/bj3350241
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