News and Publications

Regulation of Cell Function by Rho GTPases

* Hallym University, Seoul, Korea
** University of Münster, Münster, Germany

Rho GTPases control the assembly of the actin cytoskeleton, the production of reactive oxygen species, and the activity of kinase cascades that control transcription, cell growth, and cell death. This spectrum of regulatory 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 a variety of cellular, molecular, 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 Leukocyte Function

We previously established that Rac GTPase regulates the formation by human phagocytic cells of reactive oxygen species that are used to kill microbes and that result in inflammatory responses. Using a cell-free oxidase system reconstituted from recombinant protein components, we established the molecular interaction of Rac with cytochrome b independent of p67^phox, leading to a 2-step mechanism for regulation of electron transfer to form superoxide anion (Fig. 1). We also found that modulation of Rac2 GTPase activity mediates an inhibitory cross talk between leukocyte adhesion receptors and activation of NADPH oxidase. Using methods we developed to transfect primary neutrophils, we verified that Rac2 activation is rate limiting for the activation of NADPH oxidase in adherent cells. Studies to determine the biochemical basis for suppression of Rac2 activation by integrins are ongoing. These studies address the important physiologic issue of how leukocytes migrate to inflammatory sites without perpetuating continuous oxidative damage to the underlying tissue.

Regulation of RHO GTPASE

The regulatory protein GDP-dissociation inhibitor (GDI) serves as a critical control point for the function of Rho GTPases. We identified protein kinases that phosphorylate Rho GDI and specifically modulate its ability to bind Rho GTPases. This description is the first one of a selective means to release specific Rho family members from inert GDI complexes so that the GTPases can be activated by guanine nucleotide exchange factors (GEFs). Using yeast 2-hybrid screening, we identified a novel E3 ubiquitin ligase that interacts with Dbl and Vav exchange factors and that may influence the activity and localization of these Rho GEFs.

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 and motility of the cytoskeleton. We recently established a primary linkage between the actin and microtubule cytoskeletons that involves regulation of Rho GTPase via physical sequestration of Rho GEF-H1 by microtubules. We are extending studies of GEF-H1 to address the mechanisms used by cells to coordinate motile behavior and cell division. In collaborative studies with C. Waterman-Storer, Department of Cell Biology, we discovered that p21-activated kinase 1 (PAK1) is a critical regulatory link between the actin and microtubule cytoskeletons. On the basis of these findings and our research on GEF-H1, we proposed a positive-feedback model for maintenance of cell polarity (Fig. 2).

Cell Regulation By PAKS

PAKs are cellular effectors of Rac and Cdc42 GTPases. The C-terminal kinase domain of these enzymes phosphorylates substrates involved in regulating NADPH oxidase, stress responses, and the cellular actin-myosin system. The regulatory N terminus of the enzymes interacts with SH3 domain-containing proteins to modulate cytoskeletal dynamics and cell polarity. We recently established that the SH3-containing adapter Grb2 can couple PAK1 to the receptor for epidermal growth factor to mediate cytoskeletal dynamics. PAKs are important mediators of chemotaxis, wound healing, tumor metastasis, neurite outgrowth, antigen presentation, and other processes that depend on cytoskeletal polarization.

In addition to the PAK targets we previously identified (myosin light chain kinase, LIM kinase, BAD, p47^phox), we discovered new PAK-interacting substrates that may regulate additional cellular behaviors. We are using biochemical and cellular studies to characterize the regulation and biological activities of these PAK targets. The phosphorylation of cofilin, which depolymerizes and severs actin, by PAK1 bound to LIM kinase is an important regulatory point in cytoskeletal dynamics. Using a biochemical screen, we identified a unique cofilin phosphatase that may modulate the cofilin cycle through stimulus-dependent dephosphorylation of cofilin.

We also elucidated the mechanisms by which PAK activity and localization are controlled in breast cancer cells through a series of orchestrated molecular interactions involving PAK and its molecular partners PIX, PKL, and paxillin. Interestingly, PAK6, a novel kinase related to PAK, is highly expressed in prostate cancers. We determined that the kinase activity of PAK6 directly modulates signaling from the androgen receptor that is important for determining tumor aggressiveness and progression. The influence of PAK activity on the transformed phenotype of breast and prostate cancer cells is being evaluated.

Publications

Allen, M.P., Xu, M., Linseman, D.A., Pawlowski, J.E., Bokoch, G.M., Heidenreich, K.A., Wierman, M.E. Adhesion-related kinase repression of gonadotropin-releasing hormone gene expression requires Rac activation of the extracellular signal-regulated kinase pathway J. Biol. Chem. 277:38133, 2002.

Berrier, A.L., Martinez, R., Bokoch, G.M., LaFlamme, S.E. The integrin ß tail is required and sufficient to regulate adhesion signaling to Rac1. J. Cell Sci. 115(Pt. 22):4285, 2002.

Bokoch, G.M., Diebold, B.A. Current molecular models for NADPH oxidase regulation by Rac GTPase. Blood 100:2692, 2002.

Bokoch, G.M., Knaus, U.G. NADPH oxidases: not just for leukocytes anymore. Trends Biochem. Sci., in press.

Diebold, B.A., Bokoch, G.M. Regulation of the NADPH oxidase by Rac GTPase. In: G Proteins. Hamm, H. (Ed.). Academic Press, San Diego, in press. Handbook of Cell Signaling. Bradshaw, R.A., Dennis, E. (Eds.).

Gardiner, E.M., Pestonjamasp, K.N., Bohl, B.P., Chamberlain, C., Hahn, K.M., Bokoch, G.M. Spatial and temporal analysis of Rac activation during live neutrophil chemotaxis. Curr. Biol. 12:2029, 2002.

Gohla, A., Bokoch, G.M. 14-3-3 Regulates actin dynamics by stabilizing phosphorylated cofilin. Curr. Biol. 12:1704, 2002.

Li, Q., Ho, C.S., Marinescu, V., Bhatti, H., Bokoch, G.M., Ernst, S.A., Holz, R.W., Steunkel, E.L. Facilitation of Ca²⁺-dependent exocytosis by Rac1-GTPase in bovine chromaffin cells. J. Physiol. 550(Pt. 2):431, 2003.

Puto, L., Pestonjamasp, K., King, C.C., Bokoch, G.M. p21-Activated kinase 1 (PAK1) interacts with the Grb2 adapter protein to couple to growth factor signaling. J. Biol. Chem. 278:9388, 2003.

Rudrabhatla, R.S., Sukumaran, S.K., Bokoch, G.M., Prasadarao, N.V. Modulation of myosin light-chain phosphorylation by p21-activated kinase in Escherichia coli invasion of human brain microvascular endothelial cells. Infect. Immun. 71:2787, 2003.

Schrantz, N., Fowler, B., Ge, Q., Sun, Z., Bokoch, G.M. Mechanism of p21-activated kinase 6 (PAK6)-mediated inhibition of androgen receptor signaling. J. Biol. Chem., in press.

Shin, E.Y., Shin, K.S., Lee, C.S., Woo, K.N., Quan, S.H., Soung, N.K., Kim, Y.G., Cha, C.I., Kim, S.R., Park, D., Bokoch, G.M., Kim, E.G. Phosphorylation of p85 ßPIX, a Rac/Cdc42-specific guanine nucleotide exchange factor, via the Ras/ERK/PAK2 pathway is required for basic fibroblast growth factor-induced neurite outgrowth. J. Biol. Chem. 277:44417, 2002.

Wittmann, T., Bokoch, G.M., Waterman-Storer, C. Regulation of leading edge microtubule and actin dynamics downstream of Rac1. J. Cell Biol. 161:845, 2003.

Zenke, F.T., Krendel, M., King, C.C., DerMardirossian, C., Bohl, B.P., Bokoch, G.M. p21-Activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized Rho exchange factor. J. Biol. Chem., in press.

Zhou, G., Zhuo, Y., King, C.C., Fryer, B., Bokoch, G.M., Field, J. Akt phosphorylation of serine 21 on PAK1 modulates Nck binding and cell motility. Mol. Cell. Biol., in press.