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 Pl turnover. Addition of cAMP analogs restored Pl 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 Pl 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. (C) 1994 Academic Press, Inc.