Extensive Role of Unphosphorylated CREB on Gene Expression in absence of external stimuli

GRETEL KAMM; JULIETA M. CERUTI; PABLO F. SIRKIN; EDUARDO T. CANEPA

Mar del Plata, Buenos Aires

Congreso; XLIII Reunion Anual de la Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular; 2007

Sociedad Argentina de Investigacion en Bioquimica y Biologia Molecular

CB-P65. EXTENSIVEROLEOFUNPHOSPHORYLATEDCREBON GENE EXPRESSION IN ABSENCE OF EXTERNAL STIMULI KammG, Ceruti J, SirkinP, Cánepa E. Laboratorio de Biología Molecular Depto Química Biológica FCEN-UBA, Buenos Aires. E-mail: ecanepa@qb.fcen.uba.ar Transcription factor CREB induces the activity of CRE-containing gene promoters through PKA-mediated S133 phosphorylation. However, it has been reported that its binding to DNA is independent of such modification. This fact opens a question about the role, if any, of unphosphorylated CREB on gene regulation. By northern blot analysis and transfection experiments in HepG2 cells, we have demonstrated that ALAS basal transcription is diminished by CREB overexpression. This inhibition is stronger after overexpressing a CREB mutant that can not be phosphorylated or by incubating cells with the PKAinhibitor, H89. These results suggest that basal phosphorylation significantly increases the steady state level of ALAS expression. OverexpressingCREBwith a mutation in its DNA-binding domain or a CRE-mutated ALAS promoter, we demonstrated that this inhibitory effect is dependent on the CREB-binding and the presence of CRE sites on the promoter. The H-89 inhibition is released by oligodeoxynucleotide CREB decoy only when wild type CRE-ALAS promoter was used.We selected 30 hepatic genes harboring CRE regulatory regions and reported to be cAMP responsive. Interestingly, several of such genes are repressed by unphosphorylated CREB in a similar way that ALAS did. These results support a widespread role of transcription factor CREB controlling gene expression in the absence of external stimuli