Overexpresson of RIIβ, 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 RIIβ

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_α, RII_β, and RII_β-P (a RII_β, mutant at the autophosphorylation site), PKA-II levels increased whilc PKA-I levels decreased. PKA-I was almost completely eliminated in cells over expressing RII_β or RII_β-P. In contrast, over expression of either RI_α, or C_α 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_β 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 RIT, infetants were reflected in their altered phenotypic appearances. Our findings that the mutant RII_β P could not mimic the growth-inhibitory effect of RII_β P suggest the functional importance of the autophosphurylation site in RII_β, Our results suggest a role for RII_β in the suppression of neoplastic cell growth, and thus abnormal expression of R subunit isoforms of PKA may be involved in neoplastic transformation.