News and Publications

Regulation of Cell Function by Rho GTPases

G.M. Bokoch, V. Benard, T.H. Chuang, R.H. Daniels, C. DerMardirossian, B. Diebold, C.C. King, L.C. Sanders, A. Schuerman-Bartsch, F.T. Zenke, B.P. Bohl, Y. Wang

The Rho family of GTP-binding proteins (Rho, Rac, and Cdc42) has been a focus of research in our group. Rho GTPases control the assembly and activity of the actin cytoskeleton, the production of oxidants by leukocytes, the activity of regulatory kinase and phospholipase cascades, and signaling pathways that control cell growth. This spectrum of regulatory activities makes the Rho GTPases key components in such physiologic and pathologic processes as tumor growth and metastasis, wound healing, inflammatory responses, and even development. We have taken a variety of cellular, molecular, and biochemical approaches to understand how the activities of Rho GTPases are regulated, to determine the proteins these enzymes interact with to control cell function, and to ascertain how these regulatory processes are abnormal in various disease states.

CELLULAR REGULATION BY P21-ACTIVATED KINASES

While studying how Rho family GTPases regulate the phagocytic function of human leukocytes, we discovered that members of a family of serine/threonine kinases are the targets for the active forms of Rac and Cdc42. Functional and structural analysis of these enzymes, termed PAKs for p21-activated kinases, revealed their bifunctional nature. The C-terminal kinase domain phosphorylates substrates involved in regulating NADPH oxidase, stress responses, and the cellular actinomyosin system. The N terminus interacts with SH3 domain--containing proteins to modulate the actin cytoskeleton and direct polarized cell morphologies (Fig. 1). PAKs thus appear to be potentially important mediators of cell motility, wound healing, tumor metastasis, neurite outgrowth, and many other processes that depend on the dynamics of the actin cytoskeleton. Our current studies, in collaboration with U.G. Knaus, Department of Immunology, and others at TSRI, are directed at using molecular approaches to understand the function, regulation, and biology of PAKs.

RHO GTPases AS COMPONENTS IN APOPTOSIS

Cells involved in formation of tissues and organs, development of the immune system, inflammatory responses, and even abnormally growing precancerous cells are normally removed from the body by a highly regulated death response termed apoptosis. Although initiated by diverse stimuli, the death response occurs via a well-defined program involving dramatic biochemical and morphologic events that have been investigated at only a basic level. The sequence of events is as follows: The cytoplasm of the dying cells condenses toward the nucleus as the edges of the cell retract. Membrane blebbing occurs during and at the completion of the contraction phase. Finally, the cell fragments into pieces that can be effectively removed by noninflammatory phagocytic mechanisms. How this complicated series of cytoskeletal events is controlled is unknown.

We found that Rho GTPases participate in apoptosis at both biochemical and morphologic levels. Cdc42 can induce apoptotic death through its ability to stimulate the Jun/p38 kinase cascades. In collaborative studies with K. Hahn, Department of Cell Biology, we have generated Rho GTPases tagged with a fluorescent marker. These tagged enzymes are being used to determine the role of GTPases in regulating apoptotic cytoskeletal dynamics. Preliminary findings also suggest critical roles for these GTPases in controlling the cell death response.

PAK2 is a particularly important target for apoptotic proteases. It is cleaved into active N- and C-terminal pieces that can regulate aspects of the apoptotic program, including morphologic and membrane changes (Fig. 2). Continuing studies will involve determining the importance of PAK2 cleavage during apoptosis of breast cancer cells and elucidating the molecular mechanisms of action of PAKs that regulate cell death responses.

PUBLICATIONS

Ben-Ami, G., Ozeri, V., Hanski, E., Hofmann, F., Aktories, K., Hahn, K.M., Bokoch, G.M., Rosenshine, I. Agents that Inhibit Rho, Rac and Cdc42 do not block formation of actin pedestals in HeLa Cells infected with enteropathogenic Escherichia coli. Infect. Immun. 66:1755, 1998.

Bokoch, G.M. Caspase-mediated activation of PAK2 during apoptosis: Proteolytic kinase activation as a general mechanism of apoptotic signal transduction? Cell Death Diff., in press.

Bokoch, G.M., Reilly, A.M., Daniels, R.H., King, C.C., Olivera, A., Spiegel, S., Knaus, U.G. A GTPase-independent mechanism of PAK activation: Regulation by sphingosine and other biologically active lipids. J. Biol. Chem. 273:8137, 1998.

Chuang, T.H., Hahn, K.M., Lee, J.-D., Danley, D.E., Bokoch, G.M. The small GTPase Cdc42 initiates an apoptotic signaling pathway in Jurkat T lymphocytes. Mol. Biol. Cell 8:1687, 1997.

Cox, D., Chang, P., Bokoch, G.M., Greenberg, S. Requirements for both Rac and Cdc42 in membrane ruffling and phagocytosis in leukocytes. J. Exp. Med. 186:1487, 1997.

Daniels, R.H., Hall, P.S., Bokoch, G.M. Membrane targeting of p21 activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells. EMBO J. 17:754, 1998.

Dharmawardhane, S., Sanders, L.C., Martin, S.S., Daniels, R.H., Bokoch, G.M. Localization of p21-activated kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in stimulated cells. J. Cell Biol. 138:1265, 1997.

Jones, S.L., Knaus, U.G., Bokoch, G.M., Brown, E.J. Two signaling mechanisms for activation of _mß_a avidity in PMN. J. Biol. Chem. 273:10556, 1998.

Pan, Z.K., Ye, R.D., Christiansen, S.C., Jagels, M.A., Bokoch, G.M., Zuraw, B.L. Role of the Rho GTPase in bradykinin stimulated NF-B activation and interleukin-1ß gene expression in cultured human epithelial cells. J. Immunol. 160:3038, 1998.

Post, P.L., Bokoch, G.M., Mooseker, M.S. Human myosin-IXb is a mechanochemically active motor and a GAP for Rho. J. Cell Sci. 111:941, 1998.

Price, L.S., Leng, J., Schwartz, M.A., Bokoch, G.M. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol. Biol. Cell, in press.

Rudel, T., Zenke, F.T., Chuang, T.-H., Bokoch, G.M. p21-Activated kinase (PAK) is required for Fas-induced JNK activation in Jurkat cells. J. Immunol. 160:7, 1998.

Zhou, K., Pandol, S., Bokoch, G., Traynor-Kaplan, A.E. Disruption of Dictyostelium PI3K genes reduces [³²P] phosphatidylinositol 3,4 bisphosphate and [³²P] phosphatidylinositol triphosphate levels, alters F-actin distribution and impairs pinocytosis. J. Cell Sci. 111:283, 1998.

Zigmond, S.H., Joyce, M., Borleis, J., Bokoch, G.M., Devreotes, P.N. Regulation of actin polymerization in cell-free systems by GTPS and Cdc42. J. Cell Biol. 138:363, 1997.