ANALYSIS OF NOVEL PLECKSTRIN HOMOLOGY DOMAIN

超出所提供的空间。肌管蛋白（myotubullarins， MTM）属于一个脂质磷酸酶大家族，它特异性地去磷酸化磷脂酰肌醇3-磷酸（PI3P）的3'位置。MTM1在x连锁肌小管肌病中发生突变，MTMR2和MTMR13在4B型Charcot-Marie-Tooth综合征中发生突变。然而，对于MTM是如何调节的，以及不同MTM调节的特定生物过程，人们知之甚少。本提案的主要目标是研究ca激活的K通道KCa3.1 （Sk4, ikca++）的调控，我们发现该通道与MTMs的MTMR6亚家族特异性相互作用并被其抑制。在SA1中，我们将确定：(A) Kca3.1的抑制是MTMR6亚家族成员特异性的，还是其他MTMs也有抑制作用；(B) MTM6上抑制KCa3.1所需的结构域，以及这些结构域是否在其他MTMs中发挥类似的功能；(C)先前鉴定的MTMR6结合伙伴MTMR9在MTMR6抑制中的作用。为了确定MTM的CC的功能，我们将评估：(D) KCa3.1和MTMR6的CC结构域之间的相互作用是否导致磷酸酶（PT）活性的变化和/或结合是否仅将MTMR6定位在靠近KCa3.1的PM上；(E) MTMR6家族成员的CC域如何与KCa3.1和MTMR9特异性相互作用。我们将确定PI(3)P调控KCa3.1的机制。我们将确定：(A) PI3(P)对KCa3.1的调控是直接的还是间接的；(B)将测试嵌合SK3-KCa3.1通道，以确定介导PI(3)P调控的KCa3.1羧基端（CT）序列。(C) 2(B)的结果将用于指导实验，以确定KCa3.1 CT中该氨基酸序列介导PI(3)P调控的分子机制。在SA3中，我们将通过以下评估来确定MTM6作为KCa3.1负调节因子在T细胞和B细胞功能中的作用：(a) MTM6是否负调节T细胞和B细胞的增殖，如果是，通过什么机制；(B)转基因小鼠T细胞和B细胞中MTM6 （WT和磷酸酶死亡）过表达是否影响其体内功能；(C) MTM6是否调节T中枢记忆（Tcm）功能或数量。网站性能 ======================================== 节结束 ===========================================

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===========================================