Alteration of phosphoinositide metabolism by attenuation of cAMP resulting from expression of the H-ras oncogene.
Most studies characterizing H-ras have been conducted in constitutively expressing cell lines. To explore the early interaction between H-ras p21 and signal transduction systems we have utilized an NIH3T3 fibroblast line transfected with a steroid inducible MMTV H-ras vector. Exposure to dexamethasone resulted in transcription of H-ras accompanied by an increase in PI turnover. Addition of cAMP analogs restored PI metabolism to control level. We postulate that these effects are due to the regulatory action of PKA on PLC and that H-ras may interfere with cAMP metabolism, negating its regulatory effect on PLC. Therefore, we investigated the role of p21 in cAMP metabolism and PLC activity. We demonstrated that after p21 reached maximal level of expression, cAMP synthesis was reduced to 45% of the control. Radioimmunoassay of cAMP also indicated H-ras acts to inhibit adenylate cyclase activity. Further, we found a 4-fold increase in PLC activity in H-ras expressing cells that could be reversed by elevation of cAMP. Incubation with the PKA inhibitor, KT5720, resulted in activity similar to that observed in H-ras expressing cells. Additionally, we have shown no correlation between H-ras expression and GTP gamma s stimulated PI metabolism, indicating that H-ras is not functioning as a G protein in PLC activation. These results imply that H-ras functions, in this system, to decrease levels of cAMP, thus negating the regulatory effect of PKA on PLC.
Murphy
S K
SK
Department of Biological Sciences, Philadelphia College of Pharmacy and Science, PA.
Lindberg
J B
JB
Prussack
D A
DA
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D000242:Cyclic AMP / Q000378:metabolism*; D015870:Gene Expression; D011905:Genes, ras; D005822:Genetic Vectors; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008324:Mammary Tumor Virus, Mouse; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D018631:ras Proteins / Q000032:analysis / Q000096:biosynthesis*
D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D000242:Cyclic AMP; D010738:Type C Phospholipases; D018631:ras Proteins