? The Sprouty (Spry) proteins antagonize the actions of growth factors and thereby inhibit tracheal branching, angiogenesis and development of limbs and cerebellum. Among the four mammalian Sprouty (Spry) isoforms, other and we have shown that Spry1, Spry2, and Spry4 inhibit growth factor and serum induced cellular migration and proliferation. However, the precise mechanisms by which Spry proteins inhibit these processes remains to be determined. Therefore, the overall goal of this project is to identify the signaling molecules and pathways in EOF stimulated migration and proliferation of cells that are modulated by human Sprouty 2 (hSpry2). Toward this end, we have shown that hSpry2 expression increases the amount of soluble protein tyrosine phosphatase 1B (PTP1B) and decreases the particulate form of this protein. Indeed, in the presence of a dominant negative (DN) form of PTP1B or in PTP1B-/- cells the ability of hSpry2 to inhibit cell migration is attenuated; DN-PTP1B does not modify the ability of hSpry2 to inhibit cell proliferation. Interestingly, activation of Rac1 by EOF is decreased by hSpry2 and constitutively active Rac1 obliterates the ability of hSpry2 to inhibit cell migration without any effects on the ability of hSpry2 to inhibit cell proliferation. Therefore, we propose that the anti-migratory actions of hSpry2 are mediated, at least in part, by elevation in soluble PTP1B amount and activity. Since EGF-stimulated phosphorylation of p130Cas, a PTP1B substrate is decreased by hSpry2, we propose that the guanine nucleotide exchange factor activity of the p130Cas/CrkII/DOCK180/ELMO complex for Rac1 may be attenuated by hSpry2.
Thus specific aims 1 and 2 of the application will investigate the role of PTP1B, Rac1, and p130Cas in modulation of EGF-stimulated cell migration by hSpry2.
Under aim 3, we will investigate the role of PTEN in modulation of EGF-induced cell proliferation by hSpry2. These latter studies are based upon the findings that in hSpry2 expressing cells the amount of PTEN is increased and the activation of Akt by EGF is decreased. Our approaches will involve the use of cells in which one of the critical signaling elements (e.g., PTP1B or PTEN) is knocked out or in which Spry2 is knocked down by the use of siRNA and shRNA technology or conditionally knocked out. Elucidation of the mechanisms by which hSpry2 increases the amount of soluble PTP1B as well as total PTEN levels together with the identification of the signaling pathways that are modulated to antagonize growth factor-mediated activation of cell proliferation and migration will provide critical information that can be used to develop novel methods to inhibit angiogenesis, and growth of tumors as well as decrease the incidence of restenosis following vascular injury. ? ?
