Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate the 3'position of phosphatidylinositol 3-phosphate in PI3P, and play key roles in the pathogenesis of several human diseases. MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) syndrome type 4B. Little is known about the regulation of MTMs, or about which biological processes are regulated by the different MTMs. The major goals of this proposal are: (1) To identify the mechanism(s) whereby MTMR6 specifically regulates KCa3.1;and (2) To understand the role of Nucleoside Diphosphate Kinase B (NDPK-B, also known as nm23 H2), a mammalian histidine kinase, in regulating KCa3.1 channel activity and its role in CD4 T cell activation. We have evidence NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyterminus of KCa3.1. In SA1, we will determine the role and regulation of NDPK-B in T-cell receptor (TCR) signaling and T-cell activation. In (A) we will determine if NDPK-B critical for Ca2+ influx and proliferation of CD4 T cells. NDPK-B functions downstream of PI3P to activate KCa3.1. To gain insight into the function of PI3P in T cells, we will determine: (B) the mechanism whereby PI3P activates NDPK-B;and (C) how the PI3P pool that activates KCa3.1 and NDPK-B is regulated in T lymphocytes. In (D) we will use a model system to ascertain how NDPK-B, KCa3.1, MTMR6 and PI3P are regulated by TCR signaling. In SA2 we will determine (A) the role of the 2 histidine phosphatase, phosphohistidine phosphatase 1 (PHPT1) and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP), to downregulate KCa3.1 channel activity and their role in T cell activation. Our recent data strongly suggest that histidine phosphatases are negative regulators of KCa3.1. In (B) we will determine the specific role of the GRAM/PH (G/PH) domain of MTMR6 in MTMR6 targeting to the plasma membrane (PM). We now have evidence that both MTMR6's G/PH and CC domains are critical for the specific inhibition of KCa3.1 by MTMR6, acting to localize MTMR6 to the plasma membrane.
