The long-term goal of this project is to define the role of tyrosine phosphatases in glomerular cell injury. Different growth factors and cytokines are secreted or expressed in the diseased glomerulus and act on glomerular cells including mesangial cells. Signal transduction through tyrosine phosphorylation is a major pathway that mediates the biologic effects of these mediators. The level of total tyrosine phosphate content in the cell depends upon the balanced activities of protein tyrosine kinases and recently recognized protein tyrosine phosphatases (PTPases). Their plan to study molecular mechanisms of tyrosine phosphorylation/dephosphorylation that determine the activation or deactivation state of mesangial cells. They hypothesis is that, along with tyrosine kinases, alteration in the expression and/or activation of mesangial cell specific as well as other more ubiquitous tyrosine phosphatases are responsible for mesangial cell injury during renal, especially glomerular injury. They have employed a polymerase chain reaction based strategy to isolate 1.5 kb of a unique PTPase and are attempting to clone the full-length cDNA. This phosphatase will be expressed in E. coli using a plasmid expression vector under inducible conditions. PTPase will be purified and its activity will be tested and characterized. Antipeptide antibodies and antibody against bacterially produced PTPase will be raised and used to immunoprecipitate and subsequently to assay activity in mesangial cells. To understand the role of cytoplasmic and nuclear factors, they will study regulation of cloned mesangial cell specific PTPase cDNA as well as two other cytoplasmic PTPases, PTP1B and PTP1C. Role of growth factors and cytokines in regulating the activity of these phosphatases will be studied. cDNAs encoding for these PTPases will be transfected into mesangial cells and explored for their ability to activate or deactivate signals necessary for mesangial cell function. Preliminary studies in mesangial cells overexpressing PTP1C demonstrate that this PTPase enhances the proliferative potential of the cells. Regulatory effects of PTPases on signal transducing enzymes such as phospholipase Cg1, phosphatidylinositol 3 kinase, mitogen activated protein kinase and other tyrosine phosphorylated proteins will be explored. They will study a recently identified group of cytoplasmic proteins that undergo tyrosine phosphorylation and function as transcription factors for certain target genes. These proteins are called signal transducers and activators of transcription (STATs). They are tyrosine phosphorylated and show that PDGF stimulates STAT1 and also induces tyrosine phosphorylation of JAK1. Using a gel mobility shift assay, they plan to study activation of other family members of STATs in mesangial cells that overexpress PTPases. The effect of growth factors such as PDGF alone or in combination with selected relevant cytokines will also be studied. Effect of PTPases on activation of STATs and JAKs will be determined in PTPase transfected mesangial cells using immunoprecipitation and immunoblotting.
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