Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) regulates the proliferation and maturation of myeloid progenitors and enhances the function of differentiated effector cells. GM-CSF stimulation of myeloid cells results in the rapid and transient induction of the immediate early gene, egr-1, independent of protein synthesis. We previously demonstrated that the cAMP response element (CRE) contained within the -116 nucleotide egr-1 promoter region is necessary, but not sufficient, for transcriptional activation of egr-1 in GM-CSF-treated cells. We also found that the CRE-binding protein, CREB, constitutively associates with the CRE and is phosphorylated on serine 133 through a protein kinase A-independent pathway. Based on previous studies with Nerve Growth Factor-treated PC12 cells (Xing, et al. Science 273:959-963), we hypothesized that CREB is phosphorylated by pp90rsk. We identified pp90rsk in both the nucleus and cytoplasm of TF-1 cells by Western blot analysis. With GM-CSF stimulation, no increase in pp90rsk protein levels was observed. To determine whether pp90rsk phosphorylates CREB in response to GM-CSF treatment, we performed in vitro kinase assays with TF-1 cell extracts. Cells were serum- and factor-starved for 24 hours and treated with GM-CSF(1nM) for 1, 2, 5, 10, 20, 30, and 60 minutes. Extracts were incubated with pp90rsk antibody and immunoprecipitation was performed. CREB was phosphorylated within 2 to 5 minutes following GM-CSF stimulation and decreased by 30 minutes. Our results suggest that pp90rsk phosphorylates CREB during GM-CSF signal transduction. These studies provide insights into the mechanisms regulating myeloid cell proliferation and differentiation.