Scientific Report 2005

Immunology

Regulation of Cell Function by Rho GTPases

G.M. Bokoch, C. Aylott, J. Birkenfeld, M. Crawford, V. Delorme, C. DerMardirossian, A.M. DeCathlineau, B.A. Diebold, T. Huang, Y.-Y. Kao, P. Nalbant, K. Pestonjamasp, Y. Wu, T. Zhao, B.P. Bohl, A. Fowler, J. Neuberg

Rho GTPases control the assembly of the actin cytoskeleton, the production of reactive oxygen species (ROS), and the activity of kinase cascades that mediate cell growth, death, and motility. This spectrum of activities makes Rho GTPases key components of such physiologic and pathologic processes as tumor growth and metastasis, wound healing, neuronal connectivity, inflammatory responses, angiogenesis, and development. We use cellular, molecular, biophysical, and biochemical approaches to understand how the activities of Rho GTPases are regulated, to identify the proteins they interact with to control cell function, and to ascertain how these regulatory processes are abnormal in various disease states.

Rho GTPases and Human Leukocytes

We previously established that the GTPase Rac regulates the formation of ROS that are used by human phagocytic cells for microbial killing and that result in inflammatory responses. Using a cell-free recombinant system, we established the molecular interaction of Rac with cytochrome b, a component of the membrane-bound NADPH oxidase, independent of p67^phox, leading to a 2-step mechanism for regulation of electron transfer to form superoxide anion (Fig. 1). We showed that the Rac-related GTPase Cdc42 acts as a competitive inhibitor of Rac binding to cytochrome b. This inhibition results in an antagonistic cross talk between Rac and Cdc42 that modulates the formation of ROS in leukocytes and, potentially, nonphagocytic cells. This unique mechanism may coordinate formation of ROS with cytoskeletal dynamics during chemotaxis and phagocytosis.We found that an inhibitory cross talk between leukocyte adhesion receptors and NADPH oxidase activation occurs via modulation of Rac2 GTPase activity. Rac2 acts as a critical “molecular switch” that regulates formation of ROS in adherent cells. Integrin signaling inhibited activation of Vav1, the upstream guanine nucleotide exchange factor (GEF) that regulates Rac2-GTP formation and thus NADPH oxidase activity. We have now identified a mechanism by which TNF-α and certain other cytokines can overcome adhesion-induced inhibition to allow rapid formation of ROS at inflammatory sites. This mechanism requires the activity of proline-rich tyrosine kinase 2 to initiate signaling from Vav1 to Rac2. These studies (1) address the important physiologic question of how leukocytes migrate to inflammatory sites without perpetuating continuous oxidative damage to underlying tissue and (2) provide additional mechanistic insight into the inflammatory actions of TNF-α.

Fig. 1. Two-step activation mechanism for Rac GTPase–mediated regulation of oxidant formation by phagocyte NADPH oxidase.

Regulation of Rho GTPases

The regulatory protein GDP-dissociation inhibitor is a critical control point for Rho GTPase function. We are investigating the action of kinases that phosphorylate this inhibitor and modulate its ability to bind Rac GTPase. We discovered a potential positive-feedback Rac activation cycle that involves phosphorylation of Rho GDP-dissociation inhibitor by p21-activated kinase 1 (PAK1), a downstream effector of Rac and Cdc42 signaling.

We are using live-cell imaging in combination with fluorescent methods to determine the spatial and temporal localization of Rho GTPase activation. We are beginning to determine the molecular signals that govern the chemotactic responses of human leukocytes and the biochemical pathways that lead to assembly of the cytoskeleton and motility. We established a primary link between the actin and microtubule cytoskeletons that involves regulation of Rho GTPase via physical sequestration of the Rho GEF-H1 by microtubules. We recently discovered that GEF-H1 is a signaling link between microtubules in the mitotic spindle and the initiation of Rho-dependent formation of cleavage furrows in dividing cells (Fig. 2).

Fig. 2. Immunofluorescent images show colocalization of endogenous GEF-H1 with microtubules (tubulin) in the mitotic spindle.

Regulation of Innate Immunity by Anthrax Toxins

Bacillis anthracis inhibits the function of immune cells by generating lethal toxin and edema toxin. As part of a program grant funded by the Centers for Disease Control and Prevention, we are investigating the molecular basis for the suppressive effects of the anthrax toxins on human neutrophil chemotaxis and formation of ROS. A potential requirement for Rho GTPases in uptake and action of the anthrax toxins in macrophages is also under study.

Cell Regulation By PAKs

PAKs are cellular effectors of Rac and Cdc42. The C-terminal kinase domain of these enzymes phosphorylates substrates involved in regulating NADPH oxidase, stress responses, and the cellular actin-myosin system. PAKs are important mediators of chemotaxis, wound healing, tumor metastasis, neurite outgrowth, antigen presentation, and other processes that depend on cytoskeletal polarization.

The phosphorylation of cofilin, which depolymerizes and severs actin, by PAK1–LIM kinase is an important regulatory point in cytoskeletal dynamics. Using a biochemical screen, we identified a unique cofilin phosphatase, termed chronophin, that regulates stimulus-dependent activation of cofilin. Using small interfering RNA to reduce the expression of chronophin, we found that this phosphatase is involved in the control of cytokinesis during cell division. Chronophin is implicated in the formation of aneuploid cancers; it is overexpressed in such tumors and is an autoantigen in patients with cancer. We are investigating the regulation of cell motility and other cellular processes by this unique regulatory phosphatase. We are also investigating the requirement for PAK function in retrograde actin flow, a critical component of cell motility. These ongoing studies provide diverse avenues of investigation.

Publications

Birukov, K.G., Bochkov, V.N., Birukova, A.A., Kawkitinarong, K., Rios, A., Leitner, A., Verin, A.D., Bokoch, G.M., Leitinger, N., Garcia, J.G. Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac. Circ. Res. 95:892, 2004.

Bokoch, G.M. Regulation of Innate Immunity by Rho GTPases. Trends Cell Biol. 15:163, 2005.

DerMardirossian, C., Bokoch, G.M. GDIs: central regulatory molecules in Rho GTPase activation. Trends Cell Biol. 15:356, 2005.

Diebold, B.A., Bokoch, G.M. Rho GTPases and the control of the oxidative burst in polymorphonuclear leukocytes. Curr. Top. Microbiol. Immunol. 291:91, 2005.

Gohla, A., Birkenfeld, J., Bokoch, G.M. Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics. Nat. Cell Biol. 7:21, 2005.

Makino, A., Glogauer, M., Bokoch, G.M., Chien, S., Schmid-Schonbein, G.W. Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases. Am. J. Physiol. Cell Physiol. 288:C863, 2005.

Stofega, M., DerMardirossian, C., Bokoch, G.M. Affinity-based assay of Rho GTPase activation. Methods Mol. Biol., in press.

Yuan, Z.Q., Kim, D., Kaneko, S., Sussman, M., Bokoch, G.M., Kruh, G.D., Nicosia, S.V., Testa, J.R., Cheng, J.Q. ArgBP2γ interacts with Akt and p21-activated kinase-1 and promotes cell survival. J. Biol. Chem. 280:21483, 2005.

Zhao, T., Bokoch, G.M. Critical role of proline-rich tyrosine kinase 2 in reversion of the adhesion-mediated suppression of reactive oxygen species generation by human neutrophils. J. Immunol. 174:8049, 2005.