We will test the hypothesis that the tyrosine phosphorylation of cellular adaptor proteins and kinases are crucial components in the endothelin-induced intracellular signaling pathways leading to long-term effects of endothelin. We also hypothesize that protein tyrosine phosphorylation is a principal factor in evolution of cardiovascular and glomerular diseases. We believe that excessive protein tyrosine phosphorylation leading to MAPKs activation mediates inflammatory renal and cardiovascular diseases and that down-regulation of these kinases, using dual-specificity phosphatases, will have therapeutic benefit. We propose experiments to study non-receptor protein tyrosine kinases which link G-protein coupled endothelin receptors to activation of MAP kinase signaling cascades in glomerular mesangial cells and vascular smooth muscle cells. We will evaluate the roles of non-receptor tyrosine kinase Pyk2 and members of Src family of tyrosine kinases in long-term effects of ET-1. We will focus our research on regulation and role of ERK, SAP kinase and p38 kinase cascades in systems relevant for understanding the renal and vascular diseases. These studies performed in cultured mesangial and vascular smooth muscle cells will add to understanding the biological functions of MAPKs and set the stage for the development of new therapeutic strategies for vascular and renal diseases. To this end we will use the adenoviral-mediated gene transfer of different dual-specificity phosphatases into cultured cells to investigate the role of distinct MAPK signaling cascades in proliferation and/or hypertrophy of vascular smooth muscle and glomerular mesangial cells. We will evaluate the hypothesis that excessive protein tyrosine phosphorylation leading to MAPKs activation in combination with transcriptional regulation of members of MAPKs signaling cascade partially mediates inflammatory renal diseases. To evaluate the role of regulation of protein tyrosine phosphorylation in the progression of renal disease we will use two animal models of experimental renal injury. The phosphorylation status of glomerular adaptor proteins and protein kinases will be analyzed and correlated with the progression of the disease.
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