Activation of p21ras is not sufficient to ensure a complete G1 phase of the cell division cycle in mouse fibroblasts.
In the mouse BP-A31 fibroblasts, mRNAs coding the three isoforms (Ha, Ki, N) of ras are expressed, and there are no activating mutations in the codons 12, 13 or 61. We have produced a subline (Ras2) expressing an oestrogen-inducible v-Ha-ras gene. The contribution of v-Ha-ras to the overall p21ras-GTP content was evaluated by metabolic labelling with 32P. Surprisingly, p21ras-GTP complexes were predominant in the serum-deprived BP-A31 cells as well as in the Ras2 cells. The excess of p21ras-GTP was not due to the lack of the GTPase activating protein. In transient transfection experiments, the serum response element (SRE)-directed CAT was expressed in serum-deprived BP-A31 cells, and insulin caused a further two- to threefold increase in CAT activity. A dominant negative ras mutant (Ha-Ras Asn-17) cancelled both the basal and insulin-induced CAT expression in the BP-A31 but not in the Ras2 cells. Expression of v-Ha-ras in Ras2 cells did not relax their growth factor-dependence and oestradiol had only a minor mitogenic effect. We conclude that p21ras activation does not ensure a complete cell division cycle in these cells, and does not entirely account for the transduction of the mitogenic signal initiated by insulin.
Gaben
A M
AM
INSERM U55, Hôpital St Antoine, Paris, France.
Saucier
C
C
Bedin
M
M
Barbu
V
V
Courilleau
D
D
Bon-Hoa
D H
DH
Mester
J
J
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D016895:Culture Media, Serum-Free; D005347:Fibroblasts / Q000166:cytology; D016193:G1 Phase / Q000235:genetics / Q000502:physiology*; D011905:Genes, ras / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D016895:Culture Media, Serum-Free; D007328:Insulin; D012333:RNA, Messenger; D006160:Guanosine Triphosphate; D015500:Chloramphenicol O-Acetyltransferase; D015689:Oncogene Protein p21(ras)