REGULATION AND FUNCTION OF AP1 IN MATURE B CELLS

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DESCRIPTION (Adapted from the Investigator's Abstract): JunB cooperates with several different classes of transcription factors to regulate B cell activation and development and its expression is controlled at the level of transcription. This AP1 component appears to be the major such component regulating early cell cycle events after stimulation via the BCR. The current focus of the proposed work is on the regulation of junB transcription activation by BCR signals. The first goal will be to understand how a composite ets/Stat element in the junB promoter mediates BCR inducible junB transcription. Site directed mutagenesis will be used to determine the functional contribution of the individual ets and Stat motifs to BCR inducibility. EMSAs and DNA affinity chromotography will be used to identify the trans-acting factors that bind this region. An adjacent cAMP-response element (CRE) is also necessary for junB BCR-inducibility and binds CRE-binding protein 1 (CREB1). The investigator has uncovered a novel pathway for CREB1 activation by Ser133 phosphorylation. This phosphorylation is increased by the BCR via an okadaic acid-sensitive protein phosphatase (PPase) pathway. The investigator hypothesizes that this is accomplished by BCR signals inhibiting the activity of PP1 or PP-2A. Thus, the second goal of the proposal will be to test several aspects of this hypothesis using anti-PP1 and anti-PP-2A antibodies and chromatographic fractionation of B cell extracts to identify and quantitate CREB1 PPase activity. Pharmacological inhibitors and expression plasmids encoding dominant negative interfering components of BCR signaling pathways will be used to identify key intermediate proteins necessary for PPase inhibition. The data from these studies are expected to challenge existing paradigms for CREB1 regulation in mammalian cells and should elucidate a novel BCR signaling pathway and potentially new roles for ets and/or Stat binding proteins in gene regulation.