Regulation of Macropinocytosis by Hssh3bp1

描述（由申请人提供）：本提案的长期目标是了解细胞膜骨架的结构、功能和调控过程及其与人类疾病，特别是脑肿瘤发生的关系。最近的研究还涉及膜细胞骨架蛋白在正常的脑发育和行为，以及神经元的适当电生理活动。因此，这是至关重要的，以发展一个详细的了解分子和细胞的过程中调节的膜细胞骨架蛋白。膜骨架是一种多蛋白质结构，血影蛋白在其中起着主要的结构作用。基于其与α血影蛋白Src同源3结构域（SH 3）的结合特性，我们鉴定了一种新的蛋白质，命名为人血影蛋白SH 3结构域结合蛋白1（Hssh3bp1）。我们最近的工作表明，血影蛋白样蛋白和Hssh3bp1参与调节巨胞饮。巨胞饮作用是一种通过大于200 nm的囊泡发生的内吞作用，对细胞摄取液体和大分子至关重要。巨胞饮的生理意义在很大程度上是未知的，但它已被假定参与免疫反应和某些病原体的细胞摄取。值得注意的是，已经证明癌细胞系响应于生长因子刺激而上调巨胞饮作用。我们的数据表明，增加Hssh3bp1的表达抑制巨胞饮和Hssh3bp1的表达异常调节神经胶质瘤肿瘤细胞系。这些数据支持我们的工作假设，Hssh3bp1抑制巨胞饮作用，反对生长因子受体的刺激。本研究拟以人脑胶质瘤细胞系为模型系统，探讨Hssh3bp1在巨胞饮作用中的调控机制。Hssh3bp1在巨胞饮和细胞生长中的功能可能涉及其结合伴侣Abl酪氨酸激酶。我们的机制假设，这项研究的建议是，酪氨酸磷酸化Hssh3bp1抑制Abl激酶，导致细胞生长减少，由于减少磷酸化的多个有丝分裂靶点的Abl激酶。或者，Hssh3bp1与大胞饮体中的Abl结合将Abl隔离在细胞质中，因此，Abl激酶不能易位到细胞核，这是已知控制细胞生长的过程。除了测试这一假设，我们将确定是否其他蛋白质已知影响巨胞饮也调节Hssh3bp1磷酸化和控制Hssh3bp1功能。这些研究可能会导致新的信息在细胞水平上的大胞饮作用的分子调控。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the structure, function and regulatory processes of the cell membrane skeleton and its involvement in human diseases and particularly in brain tumorigenesis. Recent studies also implicate membrane cytoskeletal proteins in normal brain development and behavior, and proper electrophysiological activity of neurons. Therefore, it is critical to develop a detailed understanding of molecular and cellular processes regulated by membrane cytoskeletal proteins. Membrane skeleton is a multiprotein structure in which spectrin plays a major structural role. Based on its binding properties to alpha spectrin Src homology 3 domain (SH3) we identified a novel protein designated human spectrin S H 3 domain-binding protein 1, or Hssh3bp1. Our recent work suggest that a spectrin-like protein and Hssh3bp1 participate in regulation of macropinocytosis. Macropinocytosis is a type of endocytosis that occurs through vesicles larger than 200 nm and is critical for cellular uptake of fluid and macromolecules. Physiological significance of macropinocytosis is largely unknown but is has been postulated to be involved in immunological responses and cellular uptake of some pathogens. Significantly, cancer cell lines have been demonstrated to up-regulate macropinocytosis in response to growth factor stimulation. Our data indicate that increased Hssh3bp1 expression inhibits macropinocytosis and that Hssh3bp1 expression is abnormally regulated in glioma tumor cell lines. These data support our working hypothesis that Hssh3bp1 inhibits macropinocytosis by opposing growth factor receptor stimulation. The proposed studies focus on determining mechanism of regulatory function of Hssh3bp1 in macropinocytosis using human glioma cell lines as a model system. The function of Hssh3bp1 in macropinocytosis and in cell growth may involve its binding partner, Abl tyrosine kinase. Our mechanistic hypothesis of this research proposal is that tyrosine-phosphorylated Hssh3bp1 inhibits Abl kinase resulting in decreased cell growth due to reduced phosphorylation of multiple mitogenic targets of Abl kinase. Alternatively, Hssh3bp1 binding to Abl in macropinosomes sequesters Abl in cytoplasm, consequently, Abl kinase cannot translocate to the nucleus, a process known to control cell growth. In addition to testing this hypothesis, we will determine whether other proteins known to affect macropinocytosis also regulate Hssh3bp1 phosphorylation and control the Hssh3bp1 function. These studies are likely to lead to novel information regarding molecular regulation of macropinocytosis at the cellular level.