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.
