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2004 Scientific Report

Sites of interaction

Sites of interaction between the small GTPase RhoB and one of its effectors, mDia2, as determined by fluorescence resonance energy transfer (FRET). RhoB and mDia2 were fused to fluorescent proteins, cyan (blue-green)- and yellow-fluorescent protein, respectively. The cell was illuminated with light at a wavelength that excites the RhoB fusion protein and causes it to emit fluorescence photons; these emitted photons can cause the mDia2 fusion protein to fluoresce in turn, but only when the proteins are in very close proximity. This allows us to visualize where and when the two proteins interact. In this case, RhoB and mDia2 interact on vesicles that are important for the intracellular movement of growth factor receptors. Our data suggests that RhoB and mDia2 act as a circuit that controls the extent to which cells respond to positive growth cues. (Alberts) 8

Laboratory of Cell Structure and Signal Integration Arthur S. Alberts, Ph.D. Dr. Alberts received his Ph.D. in physiology and pharmacology at the University of California, San Diego, in 1993, where he studied with James Feramisco. From 1994 to 1997, he served as a postdoctoral fellow in Richard Treisman’s laboratory at the Imperial Cancer Research Fund in London, England. From 1997 through 1999, he was an Assistant Research Biochemist in the laboratory of Frank McCormick at the Cancer Research Institute, University of California, San Francisco. Dr. Alberts joined VARI as a Scientific Investigator in January 2000. Staff Art Alberts, Ph.D. Jun Peng, M.D. Kathryn Eisenmann, Ph.D. Lisa Alberts Laboratory Members Visiting Scientists Stephen Matheson, Ph.D. Brad Wallar, Ph.D. Students Tim Stowe Dare Odumosu Dave Beversluis Research Interests Our lab is interested in the intracellular signaling networks that regulate cell proliferation and movement and how those networks become disrupted during tumor formation. Normal cells base growth decisions upon the sum of positive and negative inputs derived from extracellular cues. These signals are processed by biochemical networks. If the networks become unbalanced—for example, by viral factors or DNA damage—the cells will arrest and/or undergo a form of programmed cell suicide in order to protect surrounding cells or tissues. In some cases, this protection system is overridden and damaged cells continue to live. As a damaged cell loses control and continues to divide unchecked, it may incur further genetic mutations that lead to tumor formation and metastasis. The objectives of the current research are to • identify key rate-limiting mechanisms that control tumor cell growth or movement and determine if they represent useful targets for anti-cancer therapy; and • develop anti-cancer drugs by exploiting our understanding of these molecular mechanisms. Our focus is the Rho family of small GTPases, their effectors, and their role in cancer pathophysiology. Rho small GTPases and malignancy Rho proteins are members of the Ras superfamily of small GTPases. Like other small GTPases, Rho proteins act as molecular switches that alternate between GTP- and GDP-bound states in which the activated (GTP-bound) proteins preferentially interact with effector proteins in the downstream network. The activated GTPases control the effectors by modulating their activities, in many cases by disrupting an autoinhibited conformation. Most of what we know about Rho proteins is from expression experiments with activated variants that lack the ability to hydrolyze GTP to GDP. Activated RhoA-G14V, for example, induces the formation of actin stress fibers; an analogous version of Cdc42 promotes the formation of microspikes and filopodia, and Rac promotes dendritic networks that form lamellae. All Rho GTPases influence the organization of the actin and microtubule networks, where the myriad signals become integrated to influence adhesion, motility, proliferation, vesicle trafficking, transcription, and survival. Some Rho family members, in particular RhoC, are overexpressed in inflammatory breast cancer and malignant melanoma, and the ability of RhoC to drive invasion and metastasis in models of those cancers cannot be matched by the closely related RhoA. On the other hand, RhoB has been hypothesized to be a negative regulator of growth and proliferation and is potentially a tumor suppressor protein. Little is known about the specific roles of the different GTPases and their respective effectors in the progression to malignancy. Diaphanous-related formins One class of GTPase effectors includes the Diaphanous-related formins (Drfs); there are three closely related Drfs in mammals: mDia1, mDia2, and mDia3. Drfs are recognized by active Rho GTPases through a loosely conserved N-termi- 9

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