The long term objective is to understand how physical factors such as electrostatics and reduction of dimensionality produce a flow of information through the calcium/phospholipid second messenger system. This proposal focuses on MARCKS, a ubiquitous major substrate of protein kinase C (PKC) that binds reversibly to calmodulin, actin, and membranes. Two hypotheses will be tested. The first hypothesis is that membrane binding of MARCKS requires both insertion of its N-terminal myristate into the interior of the bilayer and interaction of its cluster of basic residues with acidic lipids. PKC phosphorylation of serine residues weakens the electrostatic interaction and produces translocation of MARCKS from membrane to cytoplasm. The second hypothesis is that MARCKS can spontaneously self assemble into lateral domains with monovalent acidic lipids; that other, less prevalent components of this second messenger system (e.g. PKC, Src, and PIP2) will join the domains because of electrostatic considerations; and that these signal transduction domains have important physiological functions. The three specific aims are to test these hypotheses by determining how MARCKS binds to membranes, how it forms domains with acidic lipids, and the significance of these domains. Theoretical calculations using atomic models of membranes and peptides will be combined with experimental measurements to achieve these aims. Measurements of activity, fluorescence, and membrane binding will be performed using a reconstituted signal transduction system composed of phospholipid vesicles and purified proteins (heterotrimeric G protein subunits, phospholipase C, PKC, Src, and MARCKS). MARCKS is medically important because it has been implicated in secretion, cell motility, regulation of the cell cycle and transformation. The studies also are relevant to other medically important proteins that use clusters of basic residues to bind to acidic phospholipids, e.g. K-Ras 4B, Src, and HIV-1 matrix protein. Finally, preliminary data suggest that domains formed by MARCKS and the acidic lipid PIP2 produce a novel amplification of signal transduction based on the concept of positive feedback leading to an """"""""all or none"""""""" response.
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