Cell-cycle-regulated phosphorylation of cAMP response element binding protein: identification of novel phosphorylation sites

We report that the cAMP response element binding protein (CREB) undergoes c ell-cycle-regulated phosphorylation. In human amnion FL cells. CREB was exp ressed as two forms with different molecular masses, 45 and 45.5 kDa. Altho ugh asynchronous cells contained predominantly the 45 kDa forms, this form shifted to 45.5 kDa when the cells were synchronized with the early S-phase . Furthermore the expression of the 45.5 kDa band was increased when cells were treated with okadaic acid, confirming that the 45.5 kDa band was a pho sphorylated form of the 45 kDa band. Mutation analysis indicated that neith er Ser(133), the target of cAMP-dependent protein kinase and calcium calmod ulin kinase. nor Ser(129), the target of glycogen synthetase kinase 3, was responsible for the expression of the 45.5 kDa band, but that Ser(108), Ser (111) and Ser(114), located in a region matching the consensus sequence for the casein kinase II target, were required. A mutant in which Ser(111) and Ser(114) were each replaced by a glutamic residue, mimicking a phosphoryla ted state, had a higher activation potential in cAMP response element-media ted transcription. These results strongly suggest that the casein kinase II target region is involved in cell cycle-regulated phosphorylation of the C REB protein and also in transcriptional enhancement.