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 constitutivel y expressing cell lines. To explore the early interaction between H-ra s p21 and signal transduction systems we have utilized an NIH3T3 fibro blast line transfected with a steroid inducible MMTV H-ras vector. Exp osure to dexamethasone resulted in transcription of H-ras accompanied by an increase in Pl turnover. Addition of cAMP analogs restored Pl me tabolism 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 d emonstrated that after p21 reached maximal level of expression, cAMP s ynthesis was reduced to 45% of the control. Radioimmunoassay of cAMP a lso indicated H-ras acts to inhibit adenylate cyclase activity. Furthe r, we found a 4-fold increase in PLC activity in H-ras expressing cell s 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 betw een H-ras expression and GTP gamma s stimulated Pl metabolism, indicat ing that H-ras is not functioning as a G protein in PLC activation. Th ese results imply that H-ras functions, in this system, to decrease le vels of cAMP, thus negating the regulatory effect of PKA on PLC. (C) 1 994 Academic Press, Inc.