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???? 0.001. dephosphorylation of these proteins. To establish HGF as Kif2c a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an model of cultured podocytes and an model of nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity. and models of injury demonstrate that, in response to injury, recovery is initiated in an HGF-dependent manner, which involves ligand-based phosphorylation of NEPHRIN and NEPH1 leading to actin cytoskeletal reorganization and podocyte repair. Results SHP-2 is a novel binding partner for NEPH1 To identify novel NEPH1-binding proteins we performed coimmunoprecipitation experiments. The proteins that immunoprecipitated with NEPH1 were analyzed by mass spectrometry. Analysis of the Neph1-binding proteins was performed using the Scaffold proteomics software, and 123 proteins were identified. SHP-2, a product of the tyrosine-protein phosphatase non-receptor type 11 (PTPN11) gene, was one of these proteins and had previously been linked to NEPH1 (8) (Fig.?1we show that FYN kinase significantly increases NEPH1 and SHP-2 binding. To further determine if NEPH1 is a substrate for SHP-2, we tested the binding of NEPH1 with a substrate trapping SHP-2DM mutant (10, 11). SHP-2 is a phosphatase (12), and the substrate trapping SHP-2DM mutant displayed a much higher ability to bind phosphorylated NEPH1 A 438079 hydrochloride than the wildtype SHP-2 (Fig.?1were performed in triplicate, repeated three times with similar results, and representative images of the results are presented in the figure. Data are presented as mean? SEM, and 0.01, ??? 0.001, ???? 0.0001. SCR, scrambled. HGF, but not other growth factors, induces NEPH1 phosphorylation Under physiologic conditions, detection of the phosphorylated form of a protein (typically only 5%C10% of the total protein) can be challenging owing to the presence of phosphatases. Since SHP-2 appeared to be a potent phosphatase for NEPH1, we hypothesized that the phosphorylation (ligand-induced) of endogenous NEPH1 would be suppressed in the presence of SHP-2. We therefore generated stable SHP-2 knockdown (KD) human podocytes that are known to endogenously express NEPH1 and tested the level of NEPH1 phosphorylation following exposure to various growth factors using a NEPH1-specific phosphoantibody (6, 13, 14). Phosphorylation of endogenous NEPH1 was only visible in SHP-2 knockdown podocytes treated with HGF (Fig.?1and and and and 0.05, ?? 0.005, ??? 0.0005. All experiments were performed in triplicate and repeated three times with similar results, and A 438079 hydrochloride representative images of the results are presented in the figure. HGF is a novel inducer of NEPH1 and NEPHRIN phosphorylation HGF is an established activator of the mesenchymal epithelial transition (MET) receptor and SHP-2 (15, 16). Since the concept of NEPH1 and NEPHRIN phosphorylation by HGF is novel and may have significant biological and A 438079 hydrochloride clinical implications, we investigated this further using two independent techniques. First, NEPHRIN and NEPH1 were coexpressed with HGF in HEK293?cells and the cell lysates were probed using Western blot with NEPHRIN- and NEPH1-specific phosphoantibodies, which showed that NEPHRIN and NEPH1 were phosphorylated in the presence of HGF (Fig.?3test. test. ???? 0.001. All experiments were performed in triplicate and repeated three times with similar results, and representative images of the results are presented in the figure. HGF can induce phosphorylation of NEPHRIN and NEPH1 in the absence of MET Since HGF is known to be a potent activator of the MET receptor (17, 18), we investigated if MET was directly or indirectly involved in NEPHRIN and NEPH1 phosphorylation. We first used a MET receptor inhibitor (Crizotinib) (19). Crizotinib when added to NEPHRIN- and NEPH1-overexpressing HEK-293?cells was unable to attenuate the HGF-induced phosphorylation of NEPHRIN and NEPH1 (Fig.?4, and and and test. The scale bar.