Regulatory mechanism mediated by membrane lipid-protein interactions in receptor tyrosine kinase signaling

Abstract

Cell membranes are where complex molecular interactions take place that are important for cell function and regulation. Receptor tyrosine kinases (RTKs) of the cell membrane are essential components of signal transduction pathways. Research on RTK signaling has been largely focused on protein properties and interactions. Membrane lipids offer unique local environments for effective signal transduction and serve as important signaling molecules for RTKs network. Studies of molecular and spatio-temporal mechanisms for the interaction of lipids and RTK signaling component have been limited to methodological limitations. A study of signaling regulation mediated by membrane lipids will contribute to understanding the fundamentals of the RKT signaling process. Therefore, I study how membrane lipids-protein interactions in insulin receptor (IR) and epithelial growth factor receptor (EGFR) control and mediate these processes using biochemistry, molecular biology and state-of-art imaging tools. In chapter II, I dissect the molecular mechanism associated with membrane lipid-protein interaction through domain and discuss its functional significance in IR signaling. Regulation of tyrosine phosphorylation on insulin receptor substrate-1 (IRS-1) is essential for insulin signaling. The protein tyrosine phosphatase (PTP) C1-Ten has been implicated in the regulation of IRS-1, but the molecular basis of this dephosphorylation is not fully understood. Here, I demonstrate that the cellular phosphatase activity of C1-Ten on IRS-1 is mediated by the binding of the C1-Ten Src-homology 2 (SH2) domain to phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). I show that the role of C1-Ten is dependent on insulin-induced phosphoinositide 3-kinase activity. The C1-Ten SH2 domain showed strong preference and high affinity for PtdIns(3,4,5)P3. Using site-directed mutagenesis, I identified three basic residues in the C1-Ten SH2 domain that were critical for PtdIns(3,4,5)P3 binding but were not involved in phosphotyrosine binding and PTP activity. Using a PtdIns(3,4,5)P3 binding-deficient mutant, I showed that the specific binding of the C1-Ten SH2 domain to PtdIns(3,4,5)P3 allowed C1-Ten to function as a PTP in cells. Collectively, my findings suggest that the interaction between the C1-Ten SH2 domain and PtdIns(3,4,5)P3 produces a negative feedback loop of insulin signaling through IRS-1. Many open questions in biology can be tackled only if the dynamics of individual molecules can be observed noninvasively in vivo and at the appropriate spatial and temporal scale. Thus, in chapter III, to understand the functions and characteristics of membrane lipids within cells, I develop method for analyzing the single-molecule diffusional dynamics of lipids to interact with receptor in a single living cell using a homemade total internal reflection fluorescence (TIRF) microscope, referred to Lipid co-immunoimmobilization (Lipid Co-II) assay. Cholesterol is an essential constituent in the plasma membrane. Beyond its well documented effects on the physical state of the phospholipid bilayer, cholesterol has been reported to be necessary for the functional activity of many membrane receptors. The EGFR is strongly associated with cholesterol-rich subdomains of the plasma membrane, lipid rafts, which directly affect EGF-induced activation by changing the plasma membrane cholesterol content. Even though several studies have tried to explain the mechanisms by which raft disruption or movement out of raft domains increase EGFR activation, molecular mechanism of cholesterol in EGFR activation is barely understood. Here, I have investigated the single-molecule diffusional dynamics of cholesterol at first. Using Lipid Co-II assay, I observe that the cholesterol directly interacts with activated EGFR in the plasma membrane of a single living cells. I also reveal that EGFR homodimerization is critically regulated by cholesterol in a direct and a quantitative manner. This finding may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains. Also, Lipid Co-II would be a powerful tool to observe membrane lipid dynamics and to obtain new insights into the molecular mechanisms of membrane lipid-receptor interaction in the physiological condition. 60% of the drug targets are present on the cell surface and the membrane lipid and protein interactions are of increasing importance in virtually all processes. However, identification of biological significance of their interaction was limited to only a small numbered of membrane lipids and proteins. Therefore, my efforts to expand insights through the identification of biological functions (chapter II) and the development of new method (chapter III) of their interactions have significant implications. I believe that one of atlas of membrane lipid-protein interactions I found will benefit biology and medicine by shedding light on the role of membrane lipids with ‘orphan’ bioactive activity in RTK network; by deciphering novel modes of action of membrane lipids through their interactions with proteins; and by understanding their interaction in the physiological condition.