Focal adhesions (FAs) are sites where cells in culture adhere tightly to the underlying extracellular matrix (ECM). They serve to anchor bundles of actin filaments to the plasma membrane and are sites of adhesion-mediated signaling, implicated in cell migration, growth and differentiation. The assembly of FAs is regulated by the GTPase RhoA. This grant is aimed at understanding how adhesion to ECM components, such as fibronectin (FN), activates RhoA, how FAs disassemble during mitosis and in response to the development of cell-cell junctions, and how specific tyrosine phosphatases (PTPases) regulate FA assembly and function. We will explore the hypothesis that adhesion to FN activates RhoA via engagement of two types of receptor, integrins and syndecan-4, which bind to different domains of FN. We will determine whether the increase in RhoA activity results from inhibition of GTPase activating proteins (GAPs), from stimulation of guanine nucleotide exchange factors (GEFs) or from both. We will use dominant negative constructs of RhoA, as well as trapping mutants of tyrosine phosphatases to isolate RhoA GEFs downstream from integrin and syndecan-4 engagement. To examine the mitotic disassembly of FAs, we have generated an antibody that binds to the phosphorylated integrin beta 1 cytoplasmic domain. This will be used to monitor changes in integrin phosphorylation that occur during mitosis. We will measure the affinity of integrins for FN and cytoskeletal proteins during mitosis and determine whether changes in affinity are regulated by cytoplasmic domain phosphorylation. Our preliminary data reveal that the development of cell-cell junctions is associated with a sharp decrease in active RhoA. We will explore the pathway that leads to this decrease and determine whether it is responsible for the parallel loss of FAs. With respect to PTPases, we will test the hypothesis that PTPa mediates the activation of c-Src in response to integrin engagement and look for an association between this phosphatase and integrins. The PTPase, FTP-PEST has been shown to regulate cell migration and the tyrosine phosphorylation of several FA proteins. We will test the hypothesis that PTP-PEST negatively regulates the activities of both RhoA and Rac1, either by acting on tyrosine phosphorylated GEFs, such as Vav2, or by acting on FA substrates, such as p13Ocas, to disrupt their interactions.
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