ANALYSIS OF NOVEL PLECKSTRIN HOMOLOGY DOMAIN

新型 PLECKSTRIN 同源结构域分析

• 批准号: 6844881
• 负责人: EDWARD Y SKOLNIK
• 金额: $ 28.02万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-10 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6844881
• 关键词: ANALYSIS; NOVEL; PLECKSTRIN; HOMOLOGY; DOMAIN

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

超出所提供的空间。肌微管蛋白（MTM）属于一个大家族的脂质磷酸酶，其特异性地使磷脂酰肌醇3-磷酸（PI 3 P）的3'位置去磷酸化。MTM 1在X连锁肌管性肌病中突变，MTMR 2和MTMR 13在Charcot-Marie-Tooth综合征4 B型中突变。然而，关于MTM是如何调节的，或者由不同的MTM调节的特定生物过程，我们知之甚少。该提案的主要目标是研究钙激活的K通道KCa3.1（Sk 4，IKCa++）的调节，我们发现该通道与MTMR 6亚家族的MTM特异性相互作用并被其抑制。在SA 1中，我们将确定：（A）KCa 3.1的抑制是否特异于MTMR 6亚家族成员或其他MTM是否也抑制;（B）抑制KCa 3.1所需的MTM 6上的结构域和这些结构域是否在其他MTM中执行类似的功能;（C）MTMR 9（先前鉴定的MTMR 6的结合配偶体）在MTMR 6抑制中的作用。为了确定MTM的CC的功能，我们将评估：（D）KCa 3.1和MTMR 6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/或结合功能是否仅将MTMR 6定位在邻近KCa 3.1的PM处;（E）MTMR 6家族成员的CC结构域如何特异性地与KCa 3.1和MTMR 9相互作用。SA 2我们将确定PI（3）P调节KCa 3.1的机制。我们将确定：（A）PI 3（P）对KCa 3.1的调节是直接的还是间接的;（B）测试嵌合SK 3-KCa 3.1通道以确定其羧基末端（CT）中介导PI（3）P调节的KCa 3.1序列。（C）2（B）的结果然后将用于指导实验，以确定KCa3.1 CT中的该氨基酸序列介导PI调节的分子机制（3）P.在SA 3中，我们将通过以下评估来确定MTM 6作为KCa 3.1的负调节剂在T和B细胞功能中的作用：（A）MTM 6是否具有负调节T和B细胞增殖的功能，如果是，则通过何种机制;（B）MTM 6的过表达是否转基因小鼠中T和B细胞中的MTM 6（WT和磷酸酶死亡）影响它们的体内功能;（C）MTM 6是否起调节T中枢记忆（Tcm）功能或数量的作用。性能现场=