Scientific Report 2006

Immunology

Regulation of Cell Function by Rho GTPases

G.M. Bokoch, J. Birkenfeld, V. Delorme, C. DerMardirossian, A.M. DeCathelineau, D. Gianni, T. Huang, Y.-Y. Kao, J.-S. Kim, P. Nalbant, K. Pestonjamasp, S.-H. Yoon, H. Zhang, T. Zhao, B.P. Bohl, M. Crawford, B. Fowler, J.-Y. Seo, Z.-F. Chang*
* National Taiwan University, Taipei, Taiwan

Rho GTPases control the assembly of the actin and microtubule cytoskeletons, 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, 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 Rac2 regulates the formation of ROS that are used by human phagocytic leukocytes for microbial killing and that result in inflammatory responses. Our discovery of a functional interaction between Rac2 and cytochrome b, a component of the membrane-bound NADPH oxidase, independent of p67^phox, led us to propose a 2-step mechanism for regulation of electron transfer to form superoxide (Fig. 1). We are mapping the binding site for Rac2 on cytochrome b to investigate the molecular basis for regulation of ROS production by Rac2. In addition to their role in innate immunity, NADPH oxidases participate in intracellular signaling. Regulation of nonphagocytic NADPH oxidases is largely not understood, but we are investigating their modulation by kinase pathways that phosphorylate regulatory components of the oxidases.

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. Recently, we described the ability of Rac1 signaling in neutrophils to stimulate RhoA activation at the rear of cells. Such Rho GTPase cross talk promotes the development of the stable cell polarity necessary to maintain directionality of chemotaxis during inflammatory responses. Studies of the dynamics of Cdc42 activation during neutrophil chemotaxis are ongoing.

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

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 the function of human leukocytes. We have established that anthrax edema toxin and lethal toxin effectively block the ability of chemoattractant receptors to stimulate the production of ROS by human neutrophils. The molecular basis for such inhibition is currently under investigation. A requirement for Rho GTPases in the uptake and action of anthrax toxins in macrophages is also under study (Fig. 2).

Fig. 2. Rho GTPase regulation of the action of anthrax lethal toxin. Anthrax toxin is a ternary complex consisting of 1 binding subunit, protective antigen (PA), and 2 enzymatic subunits, edema factor (EF) or lethal factor (LF). Full-length PA (PA83) binds to receptors on the cell surface and is cleaved by a furinlike protease to its active form (PA63). Active PA oligimerizes, driving receptor aggregation and internalization by endocytosis. During normal maturation and acidification of the endosomes, PA forms a channel through which EF and LF are transported from the endosomal compartment and into the cytoplasm to act on their respective effectors. Rho GTPases may act to regulate endocytosis, endosomal maturation, and toxin escape or activity. Figure courtesy of Aimee DeCathelineau.

Cytoskeletal Regulation By RHO GTPases

The p21-activated kinases (PAKs) are Rac and Cdc42 effectors that serve as important mediators of chemotaxis, wound healing, tumor metastasis, neurite outgrowth, antigen presentation, and other processes dependent on cytoskeletal polarization. In collaborative studies with G. Danuser and C. Waterman-Storer, Department of Cell Biology, we are using quantitative fluorescent speckle microscopy to investigate the regulation of leading-edge actin dynamics by PAK1 downstream of Rac GTPase. We found that PAK1 plays an important role in coupling cell-edge protrusion mechanics to upstream signaling events and downstream motility.

The phosphorylation of cofilin, which depolymerizes and severs actin, by PAK1 acting through LIM kinase is an important regulatory point in cell motility. 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 discovered 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. Our recent data indicate that this unique regulatory phosphatase orchestrates actin dynamics at the leading edge by modulating cofilin activity, thereby increasing cancer cell motility stimulated by epidermal growth factor. We have also linked chronophin to cytoskeletal changes initiated during cellular energy (ATP) depletion induced by processes such as ischemia.

GDP dissociation inhibitors are critical regulators of Rho GTPase function. They have been linked to kidney disease and to the ability of cancer cells to metastasize. We found that the interaction of GDP dissociation inhibitors with Rho GTPases is regulated by phosphorylations initiated through various signaling pathways. Indeed, tyrosine phosphorylation may disrupt the regulatory capability of the inhibitors to promote cell transformation and metastasis.

Cell division also requires highly regulated actin-myosin-microtubule dynamics. We established that cross talk between the actin and microtubule cytoskeletons involving Rho regulation occurs via physical sequestration of the Rho guanine nucleotide exchange factor H1 (GEF-H1) by microtubules. GEF-H1 serves as a link between mitotic spindle microtubules and the initiation of Rho-dependent formation of cleavage furrows in dividing cells (Fig. 3). GEF-H1 activity is also controlled by cell cycle–dependent kinases. Detailed analysis of the function of GEF-H1 in cell division and motility is under way. Of interest, GEF-H1 is abundant in blood cells and is downregulated by recently developed drugs that inhibit chronic leukemias.

Fig. 3. Immunofluorescent images show colocalization of endogenous GEF-H1 with microtubules (tubulin) in the mitotic spindle.

Publications

Belvindrah, R., Nalbant, P., Ding, S., Wu, C., Bokoch, G.M., Müller, U. Integrin-linked kinase regulates Bergmann glial differentiation during cerebellar development. Mol. Cell. Neurosci. 33:109, 2006.

Birukova, A.A., Adyshev, D., Gorshkov, B., Bokoch, G.M., Birukov, K.G., Verin, A.D. GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction. Am. J. Physiol. Lung Cell Mol. Physiol. 290:L540, 2006.

Bokoch, G.M., Zhao, T. Regulation of the phagocyte NADPH oxidase by Rac GTPase. Antioxid. Redox. Signal. 8:1533, 2006.

Chang, Y.-C., Lee, H.-H., Chen, Y.-J., Bokoch, G.M., Chang, Z.-F. Contribution of guanine exchange factor H1 in phorbol ester-induced apoptosis. Cell Death Differ., in press.

Crawford, M., Aylott, C., Bourdeau, R.W., Bokoch, G.M. Bacillus anthracis toxins inhibit human neutrophil NADPH oxidase activity. J. Immunol. 176:7557, 2006.

DeCathelineau, A.M., Bokoch, G.M. Peptide inhibitors MAP the way towards fighting anthrax. Biochem. J. 395:e1, 2006.

DerMardirossian, C., Bokoch, G.M. Phosphorylation of RhoGDI by p21-activated kinase 1. Methods Enzymol. 406:80, 2006.

DerMardirossian, C., Rocklin, G., Seo, J.Y., Bokoch, G.M. Phosphorylation of RhoGDI by Src regulates Rho GTPase binding and cytosol-membrane cycling. Mol. Biol. Cell, in press.

Dong, X., Mo, Z., Bokoch, G.M., Guo, C., Li, Z., Wu, D. P-Rex1 is a primary Rac2 guanine nucleotide exchange factor in mouse neutrophils. Curr. Biol. 15:1874, 2005.

Huang, T.Y., DerMardirossian, C., Bokoch, G.M. Cofilin phosphatases and regulation of actin dynamics. Curr. Opin. Cell Biol. 18:26, 2006.

Pestonjamasp, K.N., Forster, C., Sun, C., Gardiner, E.M., Bohl, B., Weiner, O., Bokoch, G.M., Glogauer, M. Rac1 links leading edge and uropod events through Rho and myosin activation during chemotaxis. Blood. 108:2814, 2006.

Stofega, M., DerMardirossian, C., Bokoch, G.M. Affinity-based assay of Rho GTPase activation. Methods Mol. Biol. 332:269, 2006.