PH domains as Calmodulin binding domains

 描述(申请人提供)：磷脂酰肌醇3-激酶(PI-3-K，PI3K)途径传递对淋巴细胞功能至关重要的信号。PI3K在质膜上产生磷脂PIP3以招募含有Pleckstrin同源(PH)结构域的蛋白质。PH结构域是一种进化上保守的结构折叠，存在于从酵母到哺乳动物的各种生物中表达的蛋白质中。PH结构域的核心是一个由大约120个氨基酸编码的7链β桶，由两个反平行的β片层和一个C-端α螺旋组成。哺乳动物基因组编码大约300个PH结构域，在蛋白质中发现，这些蛋白质执行不同的功能，包括细胞激活、细胞骨架重组、囊泡运输和细胞周期控制。大约15%的PH结构域与高特异性和高亲和力的磷脂酰肌醇结合(KD：NM-低μM范围)。PH结构域一般通过KXn(K/R)XR基本基序中带正电荷的赖氨酸和精氨酸残基与磷脂酰肌醇相互作用。然而，并不是所有的PH结构域都与PIP3结合。几个PH结构域与选择性富含在其他膜中的磷脂酰肌醇相互作用，如高尔基膜内的PI4P或静止质膜上的PIP2。因此，将脂质特异性传递到PH结构域构成了在细胞内空间隔离不同效应蛋白的关键机制。最近，我们意外地发现钙敏感蛋白钙调蛋白是Tec、ITK和Ser/Thr激酶Akt的PIP3结合PH域的蛋白质配体。CaM和PIP3协同增强彼此与ITK PH结构域的结合，并且是T细胞最佳产生促炎细胞因子IL-17A所必需的。两个独立的PH结构域与CaM相互作用的能力使我们研究了PH结构域与CaM相互作用的可能性。在R03的应用中，我们建议系统地评估PH结构域作为CaM结合结构域，以便生成一个在线数据库，该数据库将PH结构域归类为脂质、CaM和/或小GTP酶结合。这里产生的试剂也将被存放到DNA载体库中进行一般分发。

 DESCRIPTION (provided by applicant): The phosphoinositide 3-kinase (PI 3-kinase, PI3K) pathway transduces signals critical for lymphocyte function. PI3K generates the phospholipid PIP3 at the plasma membrane to recruit proteins that contain pleckstrin homology (PH) domains. The PH domain is an evolutionarily conserved structural fold found in proteins expressed in organisms ranging from yeast to mammals. The core of the PH domain is a 7-strand β-barrel that is encoded by approximately 120 amino acids and is composed of two anti-parallel β sheets and a C-terminal α helix. The mammalian genome encodes roughly 300 PH domains found in proteins that perform diverse functions including cellular activation, cytoskeletal reorganization, vesicular trafficking, and cell cycle control. Approximately 15% of PH domains bind to phosphoinositides with high specificity and affinity (Kd: nM - low μM range). PH domains generally interact with phosphoinositides through positively charged lysine and arginine residues within the basic motif KXn(K/R)XR. However, not all PH domains bind to PIP3. Several PH domains interact with phosphoinositides that are selectively enriched in other membrane compartments, such as PI4P within the golgi membrane or PIP2 at the resting plasma membrane. Thus, conveying lipid specificity to PH domains constitutes a key mechanism for spatially sequestering distinct effector proteins within cells. Recently, we unexpectedly identified the Ca2+-sensing protein Calmodulin as a protein ligand for the PIP3-binding PH domains of the Tec kinase, Itk and the Ser/Thr kinase Akt. CaM and PIP3 cooperate to enhance each other's binding to the Itk PH domain and was required in T cells for optimal production of the pro-inflammatory cytokine IL-17A. The ability of two separate PH domains to interact with CaM led us to investigate a potential for PH domains in general to interact with CaM. In this R03 application, we propose to systematically evaluate PH domains as CaM binding domains for the purpose of generating an online database that categorizes PH domains as lipid, CaM, and/or small GTPase binders. The reagents generated here will also be deposited into a DNA vector repository for general distribution.