In this paper, we demonstrate that phosphorylation of CREB at Ser-133 is induced 64old in vivo, following treatment of PC12 cells with forskolin. By contrast, no such induction was observed in the kinase A-deficient PC12 line A126lB2 (A126). Using F9 terstocarcinoma cells, which are unresponsive to CAMP, we initiated a series of transient expression experiments to establish a causal link between phosphorylation of CREB and frans-activation of CAMP-responsive genes. Inactivating the kinase A phosphorylation site by in vitro mutagenesis of the cloned CREB cDNA at Ser-133 completely abolished CREB transcriptional activity. As CREB mutants containing acidic residues in place of the Ser-133 phosphoacceptor were also transcriptionally inactive, these results suggest that phosphorylation of CREB may stimulate transcription by a mechanism other than by simply providing negative charge.

Cyclic AMP (CAMP) mediates the hormonal stimulation of a variety of eukaryotic genes through a conserved CAMP response element (CRE)(Montminy et al., 1986; Comb et al., 1986). Transcriptional induction by CAMP is rapid, peaking at 30 min and declining gradually over 24 hr (Sasaki et al., 1984; Lewis et al., 1987). This burst in transcription is resistant to inhibitors of protein synthesis, suggesting that CAMP may stimulate gene expression by inducing the covalent modification rather than de novo synthesis of specific nuclear factors. Since all of the known cellular effects of CAMP occur via the catalytic subunit (C-subunit) of CAMP-dependent protein kinase (kinase A), it appears likely that this enzyme mediates the phosphorylation of factors that are critical for the transcriptional response. Kinase A-deficient cell lines, for example, are unable to stimulate somatostatin gene transcription in response to forskolin (Montminy et al., 1988). Furthermore, microinjection of the C-subunit into cells can directly activate CRE-dependent transcription without simultaneous addition of CAMP (Riabowol et al., 1988). We have previously characterized and isolated cDNAs for a 43 kd nuclear CRE binding protein, CREB, which binds to and stimulates transcription of the somatostatin gene in vitro (Montminy and Bilezikjian, 1987; Yamamoto et al., 1988; Gonzalez et al., 1989). Sequence analysis of the CREB cDNA predicts a single kinase A consensus