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2002 Scientific Report

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Laboratory of Cell

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. Laboratory Members Staff Jun Peng, M.D. Stephen Matheson, Ph.D. Brad Wallar, Ph.D. Akiko Vankirk, M.S. Students Nicole Neuman Dare Odomosu Research Projects Normal cells base growth decisions on the sum of positive and negative inputs derived from extracellular cues. These signals are processed by biochemical networks composed of thousands of interacting proteins and small chemicals that shuttle information from one to the other. If the system becomes unbalanced— due to the presence of viral factors or DNA damage, for example—the cells will arrest and/or undergo a form of programmed cell suicide (apoptosis) in order to protect surrounding cells or tissues. In some cases, the protection system is overridden and damaged cells continue to live. As an afflicted cell loses control and continues to divide unchecked, it may incur further mutations that lead to tumor formation and disease. Our lab is interested in the intracellular signaling networks that regulate proliferation, cell shape, and motility, as well as how cells become hijacked by disease. Our approaches depend upon a combination of molecular and cell biological techniques to define signal transduction and transformation mechanisms. In particular, we focus on the biology of the single cell and its instantaneous response to growth factor stimulation. Understanding these mechanisms is crucial in the development of anticancer treatments, because each step in a pathway may eventually be exploited for drug and gene therapy targets. The Rho family of GTP-binding proteins are signaling factors involved in cell growth responses, including changes in gene expression, cell shape, and motility. They act as molecular switch proteins: they are “on” when bound to the chemical GTP and “off” after they convert GTP to GDP. This on/off cycle is regulated by guanine-nucleotide exchange factors, or Rho GEFs. Rho GEFs are positive activators of the Rho proteins, inducing the Rho proteins to bind GTP. Once GTP is bound, Rho proteins can bind to target factors. These targets can act as Rho effectors and directly participate in signaling. Alternatively, the Rho proteins target other “switch” proteins that are part of a signaling network. The Rho proteins are from the same family of GTP-binding proteins as Ras. Ras is an oncogene mutated in many tumors. Mutant Ras protein is locked in a GTP-bound active state, but unlike Ras, similarly active GTP-bound Rho mutants are unable to transform cells. Though nontransforming, the Rho proteins are required for Ras transformation. The role that Rho proteins play in transformation is unclear, but recent work suggests that they may indirectly regulate the cell cycle. They do, however, have an important role in cancer metastases by regulating cell shape and mobility during the invasion of surrounding tissues. Oncogenic mutant Rho GEFs transform cells by essentially force-feeding GTP to Rho proteins. But unlike mutant Rho proteins locked with GTP, Rho GEFs allow cycling between GTP and GDP bound states. This cycling process appears to be key to their ability to transform cells. We are investigating this in more detail by comparing signals generated by activated Rho proteins and the oncogenic Rho 9

GEFs. On a molecular level, we are analyzing the regulation of Rho GEFs by phosphorylation, by transcriptional regulation, and by direct binding to viral and cellular proteins. The Diaphanous-related formins (DRFs) bind to activated Rho proteins. These molecular scaffolds bridge multiple growth factor–regulated signaling proteins and regulators of the cytoskeleton. One of these is Src tyrosine kinase. Src is a protooncogene whose expression is amplified in most breast tumors and whose function is critical for the growth of breast cancer cells. We have found that the DRFs bridge Src and Rho proteins in growth factor signaling. Our observation establishes an important link between two parallel signaling networks that control proliferation in response to growth factor stimulation. The DRFs are controlled by intramolecular autoinhibition, as illustrated in Figure 1. The GTPase binding domain (GBD) associates with the carboxy-terminal DAD (Dia-autoregulatory domain). This interaction is disrupted by GTPbound Rho. We are characterizing subsequent molecular events that occur as a result of Rho binding by posing the following questions: Where does DRF activation occur in cells following growth factor stimulation? Is the interaction regulated during the cell cycle or in directed cell migration? And, does Rho binding affect other DRF-binding partners activity or subcellular targeting? We are also testing the hypothesis that the DRF mDia2 binds to Src near its GTPase binding domain and that this might activate downstream signaling by disrupting autoinhibition. We are addressing these questions through a variety of methods that include digital timelapse microscopy and targeted gene disruption of the murine DRF family members in collaboration with the VARI’s Laboratory of Germline Modification headed by Pam Swiatek. Molecular regulation of the Diaphanous-related formins Rho-GDP ON Rho-GTP OFF GBD FH3 FH1 FH2 DAD Src? activation and targeting? Src WISH profilin IRSp53 actin nucleation Figure 1. Molecular regulation of the Diaphanous-related formins ? Actin remodeling machinery? External Collaborators Pierre Chardin, Institut de Pharmacologie du CNRS, Valbonne, France Phillipe Chavrier, Marie Curie Institut, Paris, France Jeff Frost, University of Texas, Houston Gregg Gundersen, Columbia University, New York George Prendergast, Lankenau Institute for Medical Research, Wynnewood, Pennsylvania Fred Wittinghofer, Max-Planck-Institut, Dortmund, Germany 10

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