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ABSTRACTcAMP, through the activation of cAMPdependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB͞ATF to cAMPresponsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RII␤ subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB͞ATF, the binding of RII␤ to a CRE was enhanced by cAMP, and in addition, RII␤ exhibited transcriptional activity as a Gal4-RII␤ fusion protein. These experiments identify RII␤ as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.cAMP-dependent protein kinase (PKA) is the major mediator of the cAMP signal transduction pathway in mammalian cells (1, 2). This enzyme consists of two catalytic (C) subunits and a regulatory (R) subunit dimer. Activation occurs when two cAMP molecules bind to each R subunit of PKA, resulting in the release of the C subunits.There are two types of PKA, type I (PKA-I) and type II (PKA-II), that share a common C subunit but contain different R subunits (RI and RII, respectively) (2). Through biochemical studies and gene cloning, four isoforms of the R subunits (RI␣, RI␤, RII␣, and RII␤) have been identified (3). Varying the ratios of two isoforms of PKA has been linked to cell growth and differentiation (4, 5). An enhanced expression of RI͞PKA-I correlates with active cell growth and cell transformation, whereas a decrease in RI͞PKA-I and an increase of RII͞PKA-II are related to growth inhibition and differentiation and͞or maturation (4, 5).Overexpression of the RII␤ subunit of PKA in several cancer cell lines results in a striking shift in PKA isozyme distribution, growth arrest, differentiation, and reverse transformation (4, 6, 7). The growth inhibition and reverse transformation correlated with nuclear translocation of RII␤, because the mutant RII␤ that failed to translocate into the nucleus was incapable of inducing reverse transformation (6).In this study, we examined whether RII␤ is a nuclear factor that can mediate cAMP responses in cAMP-responsive element (CRE)-containing genes in eukaryotic cells. Ki-rastransformed NIH 3T3 (DT) cells and DT cells infected with a retroviral vector containing the human RII␤ gene (8) (DTRII␤) or mutant RII␤-P (6) (DTRII␤-P) were used in the present study. The infectants were grown in the presence of 60 M ZnSO 4 for 48 hr before experiments to maximally induce the infected genes (6). On the basis of cell growth, ZnSO 4 treatment at 60 M for 5 days was not toxic to DT, DTRII...