The K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B, T, and mast cells. Our studies have found that T cell receptor (TCR) activation leads to activation of KCa3.1 by activating the class 2 phosphatidylinositol 3 kinase C2 Beta (PI3K-C2) leading to the generation of PI3P, which is required for the subsequent activation of a mammalian histidine kinase, Nucleoside Diphosphate Kinase B, that then activates KCa3.1. We identified the Tripartate Motif containing protein 27 (TRIM27) as a new negative regulator of PI3K-C2 and KCa3.1. TRIM27 is a member of a large family of proteins characterized by the presence of a tripartite motif, consisting of a RING finger, B box, and coiled coil (CC) domains. In this proposal, we will determine the mechanisms whereby TRIM27 regulates PI3K-C2, its physiological significance, and subsequent role as a negative regulator of KCa3.1 in lymphocyte and mast cell activation in vitro and in vivo. We have evidence that TRIM27 functions an E3 ligase and ubiquitinates and inhibits PI3K-C2. To determine the mechanism(s) whereby TRIM27 regulates PI3K-C2 and how this is affected by TCR activation, we will determine in SA1, (Ai) the type of TRIM27 mediated ubiquitination of PI3K-C2; (Aii) the lysine residues on TRIM27 and PI3K-C2 that are ubiquitinated; and (Aiii) the regions or domains on TRIM27 and PI3K-C2 that mediate their association. In 1B, we will determine: (Bi) if TRIM27 regulates TCR stimulated PI3K-C2's kinase activity and/or degradation, or whether (Bii) association of TRIM27 with PI3K-C2 is affected by TCR stimulation; (Biii) whether TRIM27 is recruited to the immunological synapse (IS), its role in IS formation and/or the recruitment PI3K-C2 to IS following TCR activation; (Biv) whether TRIM27 modulates the PI3P levels in activated T cells, or (Bv) whether TRIM27 autoubiquitination, or ubiquitination of PI3K-C2 is modulated by TCR stimulation. In (C), the role of TRIM27 stimulated SUMOylation of PI3K-C2 will be assessed. We have generated TRIM27-/- mice and have evidence that KCa3.1 channel activity and TCR- stimulated Ca2+ flux are increased in TRIM27-/- Th1 cells. In SA2 (A), we will extend these studies to other CD4 helper T cell subsets, and determine whether Th2, Th17, and Treg differentiation and/or function are altered in cells isolated from TRIM27-/- mice. In (B), we will determine the downstream signaling pathways regulated by TRIM27, and (C) whether TRIM27-/- mice are predisposed to autoimmune disease. In (D) we will undertake a nonbiased approach to identify other targets of TRIM27 ubiquitination and regulation We found that TRIM27 functions to negatively regulate FceR1 stimulated KCa3.1 channel activity and Ca2+ flux in mast cells. We will determine in SA3: (A) if PI3K-C2 mediates FceR1 stimulated activation of KCa3.1 and whether this is modulated by TRIM27; (B) whether effectors functions of TRIM27-/- mast cells are increased; (3C) the signaling pathways regulated by TRIM27 in mast cells, and/or (3D) whether TRIM27-/- mice have an increased anaphylactic response to both passive cutaneous and systemic anaphylaxis.
Negative regulators of the adaptive and innate immune system, as well as mast cells, are critical to limit inflammation and to prevent allergic responses and autoimmune disease. Failure to down regulate these responses appropriately can lead to autoimmune disease and allergy, underscoring the critical role in understanding the signaling molecules that negatively regulate these cells. We have identified TRIM27 as a new negative regulator of CD4 T and mast cells. Understanding TRIM27's role in turning off these cells is likely to shed important insights into underlying mechanisms related to autoimmune disease and allergy.
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