RECOMBINANT CYCLIC-AMP RESPONSE ELEMENT-BINDING PROTEIN (CREB) PHOSPHORYLATED ON SER-133 IS TRANSCRIPTIONALLY ACTIVE UPON ITS INTRODUCTION INTO FIBROBLAST NUCLEI

To date, it has not been possible to determine whether the single phos phorylation of the cyclic AMP response element binding factor (CREB) a t Ser-133 is sufficient for the transcriptional activation by cAMP-med iated pathways. Previous in vivo studies investigating this point have relied upon transfection of cyclic AMP-dependent kinase (cAPK) or its activation by treatment of cells with cell-permeable cAMP analogs. Ho wever, as numerous cellular proteins, including CREB, are substrates f or activated cAPK, the possibility remains that cAPK substrates other than CREB are required for the transcriptional activity of CRE-contain ing promoters. To further address this, we compared the activity of re combinant CREB phosphorylated on Ser-133 in both cell-free transcripti on assays and in vivo after introduction of the same preparations into fibroblasts by microinjection. The activity of phosphorylated CREB, n on-phosphorylated CREB, and a mutant form of CREB, containing Ala subs tituted for Ser at position 133, was found to be nearly identical in c ell-free in vitro transcription assays. In contrast, we found that onl y the phosphorylated CREB microinjected into fibroblasts resulted in t he stimulation of expression of CRE regulated genes. These results sug gest that phosphorylation of CREB on Ser-133 directly stimulates its a bility to transactivate gene expression in intact cells.