AKT induces transcriptional activity of PU.1 through phosphorylation-mediated modifications within its transactivation domain

Signal transduction by the antigen receptor complexes is critical for devel opmental progression of B-lymphocytes, which are defined by assembly and se quential expression of immunoglobulin genes, which in turn are regulated by the enhancer elements. Although proximal antigen-receptor signal transduct ion pathways are well defined, the precise nuclear factors targeted by thes e signals remained unknown. Previous studies have demonstrated that tissue- restricted transcription factors including PU.1 and PU.1 interaction partne r (PIP) function synergistically with c-Fos plus c-Jun to stimulate the kap pa E3'-enhancer in 3T3 cells. In this study, we demonstrate that the functi onal synergy between these factors is enhanced in response to mitogen-activ ated protein kinase kinase kinase, in 3T3 cells, where the enhancer is inac tive. However in S194 plasmacytoma cells, mitogen-activated protein kinase kinase kinase was able to stimulate the activity of PU.1 but unable to indu ce the kappa E3'-enhancer activity. We have found that Ras-phosphoinositide 3-kinase-dependent externally regulated kinase, AKT, induces kappa E3'-enh ancer activity in both pre-B and plasmacytoma cells. AKT stimulation of the kappa E3'-enhancer is primarily due to PU.1 induction and is independent o f PU.1 interaction with PIP. Activation of AKT had no effect on the express ion levels of PU.1 or its protein-protein interaction with PIP. Using a ser ies of deletion constructs, we have determined that the PU.1 acid-rich (ami no acids 33-74) transactivation domain is necessary for AKT-mediated induct ion. Substitution analyses within this region indicate that phosphorylation of Ser(41) is necessary to respond to AKT. Consistent with these studies, ligation of antigen receptors in A20 B cells mimics AKT activation of PU.1. Taken together, these results provide evidence that PU.1 is induced by AKT signal in a phosphoinositide 3-kinase-dependent manner, leading to inducib le or constitutive activation of its target genes.