This proposal describes experiments to continue our research on the regulation of Na+/H+ exchange activity in mitogen-stimulated, cultured human fibroblasts. The long-term goal of this project is to understand on a molecular and structural basis the mechanism by which growth factors activate the Na+/H+ exchange system. We propose specifically to: (1) Determine the kinases and phosphatases involved in the regulation of the Na+/H+ exchanger in response to mitogen stimulation. This will involve studies utilizing fusion proteins and intact cell membranes as substrates for in vitro phosphorylation assays with kinases from cell extracts and with purified kinases such as PKC, CaM kinase II and MAP kinase/MAP-like kinases. Investigation of phosphatase involvement will utilize 32P-labeled exchanger protein as a substrate for phosphatases in cell extracts. Okadaic acid will be utilized to distinguish the type of phosphatase involved; (2) Identify the site(s) of phosphorylation on the Na+/H+ exchanger molecule in mitogen-activated cells. This will involve performing in vitro phosphorylation studies utilizing as substrates fusion proteins and synthetic peptides that represent different portions of the cytosolic tail of the Na+/H+ exchanger, as well as in vivo phosphorylation experiments followed by immunoprecipitation of the exchanger with antibodies produced in our laboratory. Phosphorylation sites on the fusion proteins and the intact Na+/H+ exchanger will be identified by tryptic fragmentation of the transport protein, purification of the tryptic fragments on TLEC or reverse phase FPLC and gas phase sequence analysis of the tryptic fragments; (3) Determine whether the differential regulation of Na+/H+ exchange in HSWP and WI-38 cells (as model cells exhibiting two different types of regulation of the Na+/H+ exchanger) is the result of different regulatory pathways (kinase/phosphatase pathways) or the result of different exchanger molecules being present in the two cell types. This will involve a comparison of the phosphorylation sites and the kinase/phosphatase pathways involved in regulation of Na+/H+ exchange in these two cell types. Since the Na+/H+ exchanger is one of the best examples of a mitogen- activated membrane target whose activity is increased on growth factor stimulation, the elucidation of the molecular mechanism for the regulation of this pathway is important for our understanding of the process of normal cell growth and ultimately in the understanding of abnormal cell growth in the disease of cancer.
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