Grant: $228,000 - National Institutes of Health - May. 29, 2009
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Award Description: How variant signaling strength of the B cell antigen receptor (BCR) complex is transformed into biochemical signals is not well understood. A growing body of evidence indicates that lipid rafts function as platforms for signalling through the BCR. Bright is a B cell-restricted transcription factor that transactivates the immunoglobulin heavy chain (IgH) locus by binding to nuclear matrix attachment regions flanking the IgH intronic enhancer. Bright's DNA binding and transcriptional activities are stimulated by its direct association with Bruton's tyrosine kinase (Btk), the implicated molecule in XLA/xid. Bright undergoes a cell cycle- dependent shuttle between the nucleus and the cytoplasm and is implicated in G1/S cell cycle progression. We discovered that a small (1-2%) pool of Bright resides constitutively within lipid rafts. There it associates with Btk and the BCR signaling complex. Following BCR stimulation by surrogate antigen, Bright is discharged from rafts at a rate proportional to signal strength. An inducible association of Bright with sumoylation E2 and E3 components leads to Sumo-I-modification of Bright and its subsequent discharge from rafts into plasma membranes. BCR stimulation induces a transient, rafts-specific association and subsequent co-discharge of Bright and the F-actin linker protein Ezrin, suggesting a potential role for Bright in microfilament depolymerization - a key event in BCR signaling. This is the first functional demonstration of a transcription factor in lipid rafts. We hypothesize that Bright functions to attenuate BCR signaling; i.e., Bright-depleted signalosomes are more active than Bright-containing signalosomes. In this R21 application, we propose (1) to define the requirements for specification of Bright to lipid rafts; (2) to construct and initiate analysis of mice specifically altered for lipid-rafts-localized Bright; and (3) to identify pathways engaged by lipid rafts-localized Bright to regulate BCR signaling strength. These studies suggest another avenue for the transport of cargo between the membrane and the nucleus. They provide long-term significance for immunological tolerance, autoimmunity and immunodeficiency. Transcription factors are proteins that bind to DNA within the nucleus to initiate gene expression. We discovered that a transcription factor (Bright), which is known to possess this property, also has the unexpected property of localizing within specific structures of the cell membrane (lipid rafts). There Bright functions in an unknown manner to regulate the ability of the B lymphocyte to respond to antigen. Understanding how lipid rafts-localized Bright modulates immune responses will impact on our understanding of immunological tolerance, the immune mechanism that prevents the immune system from reacting against self; i.e., autoimmunity.
Project Description: Our R21 proposal was based on the discovery that a small (1-10%) fraction of the nucleocytoplasmic shuttling pool of Bright resides constitutively within lipid rafts prepared from transformed mature B cell lines or from primary B cell populations purified from mouse splenocytes or from human PBL. We demonstrated that in lipid rafts of resting B cells, Bright associates with Btk, TFII-I and other conventional components of the BCR signaling complex. Following BCR stimulation by surrogate antigen, Bright is discharged from rafts at a rate proportional to signal strength (anti-IgM defined as ?weak? and anti-IgM+anti-CD19 as ?strong?). Thus, we hypothesized that the lipid raft concentration of Bright is utilized for BCR threshold signaling. We have found since the grant was funded that Bright is palmitoylated at a single cysteine residue (C342), and this modification is required for its localization in lipid rafts Bright is Sumo-I conjugated on a single lysine residue (K403) at a consensus motif 401KIKKE. An inducible, rafts-specific association between Bright and Sumo-I E2/Ubc-9 and E3/PIAS-1 enzymes, along with accumulation of sumoylated Bright in the plasma membrane only after BCR stimulation, suggested that sumoylation triggered Bright discharge. That Bright self-associates into tetramers allowed us to use a dominant negative approach to test this hypothesis. We found that C342S- or C342A-Bright retroviral over-expression blocks entry into rafts and markedly enhances signaling. Retroviral over-expression of Bright mutated at either the Sumo-I consensus (401KIKK/AIAA) or the conjugated lysine (K403A) traps Bright within rafts and inhibits BCR signaling. None of these dominant negatives alter the nuclear pools, DNA binding, or IgH transactivation of Bright
Jobs Summary: The following appointments were made to this project for a total of 1.6 FTE: RES ENGR/SCI ASSOC II (0.25 FTE); RES ENGR/SCI ASSOC I (0.40 FTE); GRAD RES ASST (0.50 FTE); RESEARCH PROFESSOR (0.20 FTE); and RES ENGR/SCI ASSOC I (0.24 FTE). Calculations of Number of Jobs were made using OMB guidance. (Total jobs reported: 2)
Project Status: Less Than 50% Completed
This award's data was last updated on May. 29, 2009. Help expand these official descriptions using the wiki below.