PH domains as Calmodulin binding domains

 描述（由申请人提供）：磷酸肌醇3-激酶（PI 3-激酶，PI 3 K）通路转导淋巴细胞功能的关键信号。PI 3 K在质膜处产生磷脂PIP 3以募集含有普列克底物蛋白同源（PH）结构域的蛋白质。PH结构域是在从酵母到哺乳动物的生物体中表达的蛋白质中发现的进化上保守的结构折叠。PH结构域的核心是一个7链β桶，由大约120个氨基酸编码，由两个反向平行的β折叠和一个C末端α螺旋组成。哺乳动物基因组编码大约300个PH结构域，这些结构域存在于蛋白质中，执行多种功能，包括细胞活化、细胞骨架重组、囊泡运输和细胞周期控制。大约15%的PH结构域以高特异性和亲和力（Kd：nM -低μM范围）与磷酸肌醇结合。PH结构域通常通过碱性基序KXn（K/R）XR内的带正电荷的赖氨酸和精氨酸残基与磷酸肌醇相互作用。然而，并非所有PH结构域都与PIP 3结合。几个PH结构域与选择性富集在其他膜隔室中的磷酸肌醇相互作用，例如高尔基体膜内的PI 4P或静息质膜上的PIP 2。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，我们意外地确定了钙离子敏感蛋白钙调素作为Tec激酶，Itk和Ser/Thr激酶Akt的PIP 3结合PH结构域的蛋白质配体。CaM和PIP 3合作以增强彼此与Itk PH结构域的结合，并且在T细胞中是促炎细胞因子IL-17 A的最佳产生所需的。两个独立的PH域与钙调素相互作用的能力，使我们调查的PH域一般与钙调素相互作用的潜力。在R 03申请中，我们建议系统地评估PH结构域作为CaM结合结构域，以生成将PH结构域分类为脂质、CaM和/或小GT结合剂的在线数据库。这里产生的试剂也将存放在DNA载体库中用于一般分配。

 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.