Lorraine C. Santy Assistant Professor of Biochemistry and Molecular Biology 408 S. Frear Laboratory, University Park, PA 16802 Phone: (814) 863-6813 Fax: (814) 863-7024 E-mail: lcsanty@psu.edu B.A. in Biology, Williams College Ph.D. in Biochemistry, Harvard University Postdoctoral, University of Virginia Santy Lab Web Site

Small GTPase Regulation of Epithelial Motility

Epithelial cells are normally stationary and non-motile, however they become migratory during wound healing, tissue morphogenesis, and metastasis of epithelial tumors. Cellular migration requires precise spatial and temporal coordination of alterations in cellular signaling and architecture. Small GTPases, particularly members of the Rho family, are well-established regulators of this process. More recently a second class of small GTPases, the ADP-ribosylation factors (ARFs) have been implicated in the regulation of the actin cytoskeleton and motility. We have shown that a protein that activates the ARFs, ARNO, can play a central role in the regulation of epithelial motility. ARNO activates ARF6, and ARF6 subsequently activates phospholipase D, a lipid-modifying enzyme, and Rac1, a member of the Rho family of small GTPases. These proteins cooperate to produce the alterations in cell shape required for motility. We are continuing to investigate the regulation of ARF6 and its role in the adoption of motility by epithelial cells.

Role of Subcellular Localization of ARNO in Motility Regulation

While activation of ARF6 and Rac1 is necessary for the adoption of motility by epithelial cells, expression of activated mutants of these proteins produces ruffling but not enhanced migration. The likely explanation is that the activation of the GTPases is no longer polarized and produces a cell that ruffles and tries to move in many directions at once. Understanding the mechanisms of GTPase regulation during motility is therefore key to understanding the spatial regulation that is necessary to induce migration. The N-terminus of ARNO consists of a coiled-coil domain that mediates protein-protein interactions. We have found that a mutant of ARNO lacking this domain fails to promote epithelial motility, although it activates ARF6 as efficiently as full-length ARNO. This mutant however is deficient in inducing the activation of Rac1, suggesting that protein-protein interactions mediated by this domain are critical for ARF-induced Rac activation and motility regulation. These interactions may localize ARNO to the proper sub-cellular location or assemble a signaling complex containing ARNO and the ARF-responsive Rac-GEF. Three small scaffolding proteins have been shown to bind to the ARNO coiled-coil domain, GRASP, CYBR, and IPCEF. We are currently investigating the involvement of these proteins in ARNO-mediated regulation of epithelial motility.

Crosstalk between small GTPases

Enhancement of epithelial motility by ARNO and ARF6 requires the downstream activation of Rac1. The mechanism of this GTPase crosstalk remains poorly understood. Our recent work suggests that ARF6 induced Rac activation is mediated by the Dock180/Elmo complex. Dock180 and Elmo form a bipartite Rac-GEF that has been genetically implicated in numerous migratory events. We have found that dominant negative Dock180 and Elmo mutants inhibit ARNO induced Rac activation and motility. We are currently investigating protein complex formation between ARNO and Dock180/Elmo. We hypothesize that they may form a signaling module that serves to coordinate ARF6 and Rac activation at the leading edge of migrating cells.

ARF6 regulation in cancer cells

The initiation of metastasis requires the adoption of motility, therefore we are investigating if cancer cells exploit ARF6 activation to promote motility. We have found that levels of active ARF6 tend to be higher in more aggressive breast and colon cancer cell lines. We are investigating the mechanism of this enhanced ARF6 activity and if it contributes to the enhanced motility of these cell lines.

Representative Publications:

• Santy, L. C., Ravichandran, K. S., and Casanova, J. E. (2005) “The DOCK180/Elmo complex couples ARNO-mediated Arf6 activation to the downstream activation of Rac1” Current Biology, in press.
• Martinu, L, Masuda-Robens, J. M., Robertson, S. E.,Santy, L. C., Casanova, J. E., and Chou, M. M. (2004) “The TBC domain protein TRE17 regulates plasma membrane-endosomal trafficking through activation of Arf6.” Molecular and Cellular Biology,24: 9752-62.
• Santy, L. C. (2002) “Characterization of a Fast Cycling ADP-Ribosylation Factor 6 Mutant” J. Biol. Chem. 277: 40185-40188.
• Santy, L. C. and Casanova, J. E. (2002) “GTPase Signaling: Bridging the GAP between ARF and Rho” Dispatch, Curr. Biol. 12: R360-R362.
• Santy, L. C. and Casanova, J. E. (2001) “Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D.” J. Cell Biol. 154: 599-610.
• Santy, L. C.., Frank, S. R., and Casanova, J.E. (2001) “Expression and Analysis of ARNO and ARNO Mutants and Their Effects on ADP-Ribosylation Factor (ARF)-Mediated Actin Cytoskeletal Rearrangements.” Meth. Enzymol. 329: 256-64.

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