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
Sj. Fuller et al., 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, Biochemical journal, 335, 1998, pp. 241-246
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)-a
lpha-actin. To determine which signalling pathways mediate these respo
nses, myocytes were transfected with oncogenic (V12) Ras mutants which
interact selectively with different effecters and their effects on lu
ciferase (LUX) reporter plasmids were examined. V12 human Ras (V12HRas
), itself, activated ANF-LUX 9.6-fold, whereas mutants of V12HRas, whi
ch selectively stimulate Ral guanine nucleotide dissociation stimulato
r (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 respect
ively. 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-fol
d by V12HRas, E37G, D38E and Y40C respectively, and a similar pattern
of activation was also observed using a c-fas serum-response element-L
UX 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 (p110K22
7E) stimulated ANF-LUX, SkM-alpha-actin-LUX, c-fos-serum-response elem
ent-LUX and Rous sarcoma virus-LUX by 3.1-, 3.2-, 2.1- and 2.9-fold re
spectively, but the co-transfected cytomegalovirus-beta-galactosidase
reporter gene was activated to a similar extent (1.9-fold). These resu
lts suggest that Raf, Ral.GDS and PI-3-K can all transduce transcripti
onal responses to V12HRas, but that the specific induction of genes as
sociated with the hypertrophic response is not mediated through PI-3-K
.