-EJB 95 l670/1 I S 1 74T human colon carcinoma cells that contain approximately equal amounts of CAMP-dependent protein kinase (PKA) isozymes, PKA-I and PKA-11, were infecled with retroviral vectors coding for regulatory (R) and catalytic (C) subunits of human PKA. In cells overexpressing RIIcr, Rllij, and RTT, P (a RII,, mutant at the autophosphorylation site), PKA-JT levels increased whilc PKA-I levels decreased. PKA-I was almost completely eliminated in cells overexpressing RII,{ or RIT,,-P. In contrast, overexpression of either RI,, or C, had little or no effect on PKA isozyme levels. Although all infectants expressed high levels of PKA subunit inRNAs in accordance with gene introduction, the R subunit protein expression was reflected in PKA isozyme levels rather than in subunit mRNA levels. Only RII, infectants demonstrated marked growth inhibition in monolayer C U~~L I I .~, reduced thymidine incorporiition into DNA, and inability to grow in semisolid medium or in serum-free medium. Conversely, it11 other infectants displayed growth propcrties similar to uninfected parental cells. The growth-retardation properties of RIT, infectants were reflected i n their altered phenotypic appearances. Our findings that the mutant RIl,rP could not mimic the growth-inhibitory effect of RII, suggest thc functional importance of the autophosphurylation site in RII,,. Our results suggest a role for KIT,, in the suppression of neoplastic cell growth, and thus abnormal expression of R subunit isoforms of PKA may bc involved in neoplastic transformation.