The ability of kidney epithelial cells to regulate their morphology is critical for developmental nephrogenesis and tubular repair following renal injury, suggesting that understanding how epithelial cell morphologic events are regulated will provide us with unique tools for enhancing recovery from acute renal failure and potentially modifying the course of chronic renal tubular diseases. This proposal focuses on a newly discovered cytosolic signaling protein, Gab 1, which has been found to associate in a regulated fashion with the receptors known to mediate morphogenesis and to directly initiate renal epithelial cell tubulogenesis in cell culture. We have found that one region of Gab1, the met-binding domain (MBD), can associate in a phosphorylation- specific manner with both ERK2 and ERK1, downstream effectors of the MAPK signaling cascade. We have further found that an inhibitor of MAPK signaling dramatically inhibits renal epithelial cell migration and branching morphogenesis. Thus, we believe that a novel interaction between Gabl and the MAPK signaling cascade regulates epithelial morphogenesis. We propose to investigate this hypothesis by first utilizying bacterially expressed Gab1 met binding domain to determine how ERK phosphorylation regulates the MBD-ERK association (Specific Aim la). A degenerate phosphopeptide library will be utilized to examine the possibility that the MBD represents a novel phosphoamino acid binding domain for regulating signaling protein interactions, and to determine the preferred MBD-binding sequence and thus potentially identify novel target signaling proteins for Gabl binding (Specific Aim lb). The regulation of the MBD-ERK2 interaction in the intact cell will next be examined by determining how growth factor addition alters Gabl-ERK2 association (Specific Aim 1c). The results of Specific Aim 1 will then be used to determine how the Gabl-MAPK interaction affects cell signaling. The ability of Gabl to regulate ERK2 kinase activity and/or specificity will be examined (Specific Aim 2a), as will the possibility that Gab1 phosphorylation by ERK2 regulates Gab1 mediated signaling protein interactions (Specific Aim 2b). Finally, the phenotypic outcome of the regulation of signaling proteins by the Gab1-ERK interaction as identified in Specific Aim 2 will be investigated utilyzing the cell lines developed in Specific Aims 1 and 2 to perform cell migration and tubulogenesis assays aimed at determining how these signaling cascades mediate renal epithelial morphogenesis (Specific Aim 3).
