REGULATION OF SOMATOSTATIN GENE-TRANSCRIPTION BY CYCLIC ADENOSINE-MONOPHOSPHATE

Cyclic adenosine monophosphate (cAMP) stimulates transcription of soma tostatin and other target genes with burst-attenuation kinetics. The k inetics of protein kinase (PK-A)-dependent cAMP response element bindi ng protein (CREB) phosphorylation closely parallel the changes in tran scription of cAMP-responsive genes by run-on assay. Nuclear translocat ion of PK-A, visualized by microinjection of fluorescently labeled PK- A holoenzyme, appears to represent the rate-limiting step in CREB phos phorylation and transcriptional activation. We and others have recentl y characterized a CREB-binding protein (CBP), which specifically recog nizes sequences within the Ser133 phosphorylated form of CREB. CBP doe s not regulate the DNA binding, dimerization, or nuclear targeting pro perties of CREB, but binds selectively to the kinase-inducible 60 amin o acid trans-activation domain (KID) of CREB, critical for PK-A-induci ble transcription. We developed an antiserum directed against amino ac id 634-648 within the CREB-binding domain of CBP. We detected a 265-kd polypeptide by Western blot as predicted from the cDNA, which coincid ed with the predominant phospho-CREB-binding activity in Hela nuclear extracts by ''Far Western'' blot assay. An identical phospho-CREB-bind ing activity was also found in NIH-3T3 cells. This phospho-CREB-bindin g protein appeared to be specific for Ser133-phosphorylated CREB, beca use no such band was detected with CREB labeled to the same specific a ctivity at a nonregulatory phosphoacceptor site (Ser156) by casein kin ase II (CKII). Following microinjection into nuclei of NIH-3T3 cells, a cAMP response element (CRE)-lacZ reporter was markedly induced by tr eatment with 8-Br cAMP plus isobutyl methyl xanthine (IBMX). Coinjecti on of CBP antiserum with the CRE-lacZ plasmid inhibited cAMP-dependent activity in a dose-dependent manner, but control immunoglobulin G (Ig G) had no effect on this response. We can now begin reconstituting PK- A-dependent transcription in vitro, using well-characterized proteins such as CREB, TAF 110, and CSP. The assembly of such factors on cAMP-r egulated promoters like somatostatin may enable responsiveness to a va riety of hormonal stimuli that employ cAMP as their second messenger. Copyright (C) 1996 by W.B. Saunders Company