EXPRESSION OF A PEPTIDE INHIBITOR OF PROTEIN PHOSPHATASE-1 INCREASES PHOSPHORYLATION AND ACTIVITY OF CREB IN NIH 3T3 FIBROBLASTS

We have examined the activity and phosphorylation state of the cyclic AMP (cAMP) response element binding factor (CREB) in intact NIH 3T3 ce lls following microinjection of expression plasmids encoding regulator y proteins of type 1 (PP1) and 2A (PP2A) serine/threonine-specific pro tein phosphatases. Changes in CREB phosphorylation in the injected cel ls were monitored by indirect immunofluorescence using an affinity-pur ified antiserum (Ab5322) which specifically recognizes CREB phosphoryl ated at Ser-133, and changes in transcriptional activity of CREB were monitored by expression of a reporter gene regulated by cAMP. cAMP-sti mulated phosphorylation in NIH 3T3 cells is normally transient, and as expected, after stimulation of cells with cell-permeable cAMP analogs , the level of phosphorylated CREB was found to initially increase and then return to a basal level within 4 h. Microinjection of an express ion vector encoding a constitutively active form of inhibitor 1 (I-1), a PP1-specific inhibitor, by itself resulted in an apparent increase in phosphorylated CREB in unstimulated tells. Moreover, injection of t he I-1 vector resulted in the prolonged appearance of phosphorylated C REB in cells after cAMP stimulation. In contrast, injection of a plasm id encoding simian virus 40 small t antigen, which interacts with PP2A to inhibit its activity towards several phosphoprotein substrates, ha d no effect on the phosphorylation state of CREB in stimulated or unst imulated NIH 3T3 cells. Consistent with these results, injection of th e I-1 expression vector activated expression from a coinjected CRE-lac Z reporter plasmid, indicating that the increased phosphorylation of C REB also activated its transcriptional activity. These results provide further evidence for a role of a PP1 as the primary protein (Ser/Thr) phosphatase regulating the dephosphorylation of Ser-133 and thereby l imiting the transcriptional activity of CREB.