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We have examined the activity and phosphorylation state of the cyclic AMP (cAMP) response element binding factor (CREB) in intact NIH 3T3 cells following microinjection of expression plasmids encoding regulatory proteins of type 1 (PP1) and 2A (PP2A) serine/threonine-specific protein phosphatases. Changes in CREB phosphorylation in the injected cells were monitored by indirect immunofluorescence using an affinity-purified antiserum (Ab5322) which specifically recognizes CREB phosphorylated at Ser-133, and changes in transcriptional activity of CREB were monitored by expression of a reporter gene regulated by cAMP. cAMP-stimulated 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 expression 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 cells. Moreover, injection of the I-1 vector resulted in the prolonged appearance of phosphorylated CREB in cells after cAMP stimulation. In contrast, injection of a plasmid encoding simian virus 40 small t antigen, which interacts with PP2A to inhibit its activity towards several phosphoprotein substrates, had no effect on the phosphorylation state of CREB in stimulated or unstimulated NIH 3T3 cells. Consistent with these results, injection of the I-1 expression vector activated expression from a coinjected CRE-lacZ reporter plasmid, indicating that the increased phosphorylation of CREB 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 limiting the transcriptional activity of CREB.