Overexpresson of RII(beta) regulatory subunit of protein kinase A in human colon carcinoma cell induces growth arrest and phenotypic changes that are abolished by site-directed mutation of RII(beta)

LS-174T human colon carcinoma cells that contain approximately equal amounts of cAMP-dependent protein kinase (PKA) isozymes, PKA-I and PKA-II, were infected with retroviral vectors coding for regulatory (R) and catalytic (C) subunits of human PKA. In cells overexpressing RII(mu) RII(beta), and RII(beta)-P (a RII(beta) mutant at the autophosphorylation site), PKA-II levels increased while PKA-I levels decreased. PKA-I was almost completely eliminated in cells overexpressing RII(beta) or RII(beta)-P. In contrast, overexpression of either RI(alpha) or C-alpha had little or no effect on PKA isozyme levels. Although all infectants expressed high levels of PKA subunit mRNAs in accordance with gene introduction, the R subunit protein expression was reflected in PKA isozyme levels rather than in subunit mRNA levels. Only RII(beta) infectants demonstrated marked growth inhibition in monolayer culture, reduced thymidine incorporation into DNA, and inability to grow in semisolid medium or in serum-free medium. Conversely, all other infectants displayed growth properties similar to uninfected parental cells. The growth-retardation properties of RII(beta) infectants were reflected in their altered phenotypic appearances. Our findings that the mutant RII(beta)-P could not mimic the growth-inhibitory effect of RII(beta) suggest the functional importance of the autophosphorylation site in RII(beta). Our results suggest a role for RII(beta) in the suppression of neoplastic cell growth, and thus abnormal expression of R subunit isoforms of PKA may be involved in neoplastic transformation.