SIGNAL TRANSDUCTION VIA RHO GTPases

The control and integration of cellular activities initiated by external signals involves GTP-binding proteins (G proteins or GTPases) that use the binding and hydrolysis of GTP as a means to regulate macromolecular interactions and activities. Our goal is to determine how Rho GTPases function at the molecular level, and how they are in turn modulated by regulatory molecules which control their activity. Our laboratory utilizes diverse biochemical, cellular, biophysical, and molecular biological approaches to address these questions.

Rho family GTPases regulate cellular processes particularly important for immune cell function. We are characterizing the signaling mechanisms controlling activation of Rho GTPases and studying the target proteins which mediate their distinct abilities to regulate the actin and microtubule cytoskeletons in motile cells, the human neutrophil NADPH oxidase used for bacterial killing, and leukocyte chemotaxis. We are proceeding by a) biochemical characterization of regulatory pathways and proteins, b) use of dominant stimulatory and inhibitory proteins and siRNA-mediated protein depletion to analyze cytoskeletal and enzymatic changes both in vivo and in vitro, and c) through the use of protein transduction systems which allow us to efficiently introduce proteins into human leukocytes.  Our studies on the regulation of the phagocyte NADPH oxidase by Rac2 GTPase have led to new insights into the regulation of this important component of inflammation and innate immunity.

Research in our laboratory on p21-activated kinase (PAK) has implicated this Rac/Cdc42 target in control of polarized actin assembly, including neurite extension in PC12 cells and pseudopod formation in human neutrophils, in apoptotic signaling and stress responses in lymphocytes, and in transcriptional/cell cycle control. In addition to regulation by GTPases, we showed PAK activity to also be controlled by membrane targeting and sphingolipids. We are currently utilizing live cell imaging and fluorescence-based technologies to investigate the biology and spatial organization of Rho GTPases and PAKs in intact cells. In-depth studies of the PAK-regulated cofilin/ADF cycle that controls actin dynamics have led to the identification of a novel cofilin/ADF phosphatase regulating cell motility and cytokinesis. Utilizing quantitative fluorescent speckle microscopy, we have now shown PAK1, acting through cofilin, to serve as a key regulatory point for the organization and activities of the two functionally distinct actin networks at the leading edge of cells. 

The proteins regulating Rho GTPase activity play important roles themselves in both normal physiological processes and in disease.  We identified the oncogenic guanine nucleotide exchange factor GEF-H1 as a PAK substrate which couples Rho GTPase activation to microtubule dynamics. We have now shown GEF-H1 to be a key regulator of cell division. Using live cell imaging of Rho activity, we established GEF-H1 to regulate the activation of RhoA during cytokinesis.  RhoGDI and D4GDI are also important regulators of Rho GTPase activation and membrane-cytosol cycling.  We showed RhoGDI and D4 GDI to be substrates for regulatory kinases, including Src family kinases.  Tyrosine phosphorylation of RhoGDI inhibits the recycling of active Rho GTPases back to the cytosol, prolonging their interaction with effectors and potentially contributing to the action of Src in cancers, etc.  Our ongoing studies provide new insights into the control of basic biological processes in immune and other cells whose regulation may be abnormal in disease states.