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A nuclear protein, CREB, has been isolated from rat brain and shown to stimulate transcription of the cyclic AMP-responsive gene somatostatin as a dimer. Biochemical analysis suggests that dimerization and transcriptional efficacy of CREB protein in vitro are regulated by phosphorylation. These findings demonstrate that cellular signals can modulate gene expression by regulating the covalent modification of pre-existing nuclear factors.

Cyclic AMP regulates the expression of a number of genes through a conserved promoter element, the CRE1. Moreover, transcriptional induction by cAMP requires the activation of cAMP-dependent protein kinase (protein kinase A). We have previously characterized the cAMP response element binding protein (CREB) in PC12 cells and brain tissue as a nuclear factor, of relative molecular mass 43,000, whose transcriptional efficacy is regulated by protein kinase A phosphorylation. CREB stimulates transcription on binding to the CRE as a dimer. Experiments suggesting that the dimerization and transcriptional efficacy of CREB are each stimulated by phosphorylation at distinct sites prompted us to suggest that CREB is regulated by multiple kinases in vivo. We now report the isolation of a cDNA clone for rat CREB using amino-acid sequence information from purified CREB protein. Sequence analysis of this CREB cDNA predicts a cluster of protein kinase A, protein kinase C and casein kinase II consensus recognition sites near the N terminus of the protein. The proximity of these potential phosphorylation sites to one another indicates that they may interact either positively or negatively to regulate CREB bioactivity.

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Cyclic AMP mediates the hormonal stimulation of a number of eukaryotic genes by directing the protein kinase A (PK-A)-dependent phosphorylation of transcription factor CREB. We have previously determined that although phosphorylation at Ser-133 is critical for induction, this site does not appear to participate directly in transactivation. To test the hypothesis that CREB ultimately activates transcription through domains that are distinct from the PK-A site, we constructed a series of CREB mutants and evaluated them by transient assays in F9 teratocarcinoma cells. Remarkably, a glutamine-rich region near the N terminus appeared to be important for PK-A-mediated induction of CREB since removal of this domain caused a marked reduction in CREB activity. A second region consisting of a short acidic motif (DLSSD) C terminal to the PK-A site also appeared to synergize with the phosphorylation motif to permit transcriptional activation. Biochemical experiments with purified recombinant CREB protein further demonstrate that the transactivation domain is more sensitive to trypsin digestion than are the DNA-binding and dimerization domains, suggesting that the activator region may be structured to permit interactions with other proteins in the RNA polymerase II complex.A number of growth factors and hormones regulate the expression of target genes by stimulating the phosphorylation of specific transcription factors. We have previously characterized the nuclear factor CREB, for example, which stimulates transcription of genes in response to the secondmessenger cyclic AMP (cAMP) (5,9,11,17). Biochemical and in vitro mutagenesis experiments have revealed that CREB is activated by phosphorylation at a single protein kinase A (PK-A) phosphoacceptor, site Ser-133 (4). Since phosphorylation at Ser-133 activates transcription without changing DNA-binding affinity, it appears that phosphorylation of CREB directly modulates the efficacy of the transactivation domain.Previous work showing that CREB can stimulate transcription of previously unresponsive genes when a cAMP response element (CRE) (11) is attached to these promoters has prompted us to hypothesize a general activating motif in CREB that would interact with ubiquitous proteins in the RNA polymerase II transcription complex. Indeed, two such general activating motifs have been described in a number of nuclear factors, one glutamine rich and the other containing acidic residues (1, 13). Nevertheless, a number of nuclear factors have activation domains that do not fit into either category. The direct increase in negative charge accompanying phosphorylation suggested that proteins like CREB would stimulate transcription by providing an acidic surface. The inability of acidic residues to substitute for the serine phosphoacceptor, however, has argued against this model. Rather, by analogy with other kinase substrates, phosphorylation might activate CREB by triggering a conformational change in the protein which, in turn, would permit a second activating group in the protein to ...