ANALYSIS OF NOVEL PLECKSTRIN HOMOLOGY DOMAIN

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EXCEED THE SPACE PROVIDED. Myotubularins (MTM) belong to a large family of lipid phosphatases that specifically dephosphorylate the 3' position of phosphatidylinositol 3-phosphate (PI3P). MTM1 is mutated in X-linked myotubular myopathy and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome, type 4B. However, little is known about how MTMs are regulated, or the specific biological processes regulated by the different MTM. The major goal of this proposal is to study the regulation of a Ca-activated K channel, KCa3.1 (Sk4,IKCa++) that we found specifically interacts with and is inhibited by the MTMR6 subfamily of MTMs. In SA1 we will determine: (A) whether inhibition of Kca3.1 is specific to MTMR6 subfamily members or whether other MTMs also inhibit; (B) the domains on MTM6 required to inhibit KCa3.1 and whether these domains perform similar functions in other MTMs; (C) The role of MTMR9, a previously identified binding partner for MTMR6, in MTMR6 inhibition. To determine the function of MTM's CC we will assess: (D) whether interaction between the CC domains of KCa3.1 and MTMR6 leads to changes in phosphatase (PT) activity and/or whether binding functions only to localize MTMR6 at the PM adjacent to KCa3.1; (E) how CC domains of MTMR6 family members specifically interact with KCa3.1 and MTMR9. SA2 we will determine the mechanism by which PI(3)P regulates KCa3.1. We will determine: (A) whether regulation of KCa3.1 by PI3(P) is direct or indirect; (B) chimeric SK3-KCa3.1 channels will be tested to determine the KCa3.1 sequence in its carboxy-terminus (CT) that mediates regulation by PI(3)P. (C) The results from 2(B) will then be used to guide experiments to identify the molecular mechanism whereby this amino acid sequence in the KCa3.1 CT mediates regulation by PI(3)P. In SA3 we will determine the role MTM6 as a negative regulator of KCa3.1 in T and B cell function by assessing: (A) whether MTM6 functions to negatively regulate T and B cell proliferation and, if so by what mechanism; (B) whether overexpression of MTM6 (WT and phosphatase-dead) in T and B cells in transgenic mice affects their in vivo function; (C) whether MTM6 functions to regulate T central memory (Tcm) function or number. PERFORMANCE SITE ========================================Section End===========================================