Professor of Biochemistry and Biophysics
PHD - Yale University
HONORS & AWARDS
- Phillip & Ruth Hettleman Prize - 2001
- Jefferson Pilot Award - 1998
Ras and Rho GTPases, Their Modulators and Effectors
The Ras proteins are members of a large superfamily of Ras-related proteins that are key regulators of signal transduction pathways that control normal cell growth. Mutated Ras proteins are found in 30% of human cancers and promote uncontrolled cell growth, invasion, and metastasis. One major branch of the Ras superfamily consists of members of the Rho GTPases (e.g., RhoA, Rac1, Cdc42). Like Ras, Rho GTPases also serve as on-off switches to relay extracellular signal-mediated stimuli to cytoplasmic signaling pathways including those involved in cellular growth control. Distinct from Ras, however, Rho GTPase-mediated signaling pathways modulate cell morphology and actin cytoskeletal organization.
Our most recent work has elucidated a novel mechanism by which monoubiquitination upregulates Ras activity. Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to hyper-activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitination has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation. We are following up on these initial studies to better understand the molecular basis for how ubiquitin ligation regulates Ras conformational dynamics, GTP downregulation by GAP proteins and effector binding. We are also investigating the isoform dependence of ubiquitination and its role in Ras-mediated tumorigenesis, in collaboration with Atsuo Sasaki (U. of Cincinnati) and Channing Der (UNC-CH).
Once activated, Ras can regulate numerous and complex pathways that control cellular growth. However, the activation state of Ras was previously thought to be regulated solely by protein modulatory factors. We are finding that the regulation is more complicated than previously envisioned. In addition to ubiquitination, reactive oxygen or nitrogen species can also regulate the activity of Ras and Rho GTPases. We are currently characterizing mechanisms of regulation by oxidative thiol modification.
Current research projects in the Campbell laboratory include: characterization of post-translational modification of Ras and Rho GTPases by monoubiquitination and redox regulation using structural, biophysical and biochemical studies; characterization of small molecule Ras inhibitors; identification, characterization and structural elucidation of factors that act on Ras, Rap and Rho family GTPase proteins.
Cell Adhesion Molecules: Focal Adhesion Kinase, Vinculin and Palladin
Our work on Rho related GTPases, has led us into the area of focal adhesion assembly and integrin-mediated signaling, as the RhoA GTPase is involved in assembly of focal adhesions, whereas Rac and Cdc42 affect the organization of the actin cytoskeleton and regulate cell migration. Our studies have focused on the cell adhesion proteins, Focal Adhesion Kinase (FAK) and vinculin and palladin.
Focal Adhesion Kinase (FAK) is a 125 kDa protein that co-localizes with integrins at focal adhesions upon cell adhesion to the extracellular matrix. FAK is involved in a multiple cell signaling pathways that include regulation of cell motility and cell survival. In addition, FAK may function in the pathology of human cancer including prostate, colon and breast cancers. The focal adhesion targeting (FAT) domain is located at the carboxy-terminus of FAK (residues 920-1053) and is critical for recruitment of FAK to focal adhesions and subsequent activation. We solved the NMR solution structure of the FAT domain of FAK and well as the FAT domain complexed to a paxillin derived peptide. NMR in combination with isothermal titration calorimetry studies have helped to delineate the molecular and thermodynamic basis of paxillin interactions with FAT. Current studies are centered on the role of FAT domain phosphorylation and FAT domain conformational dynamics in FAK function.
In addition to our investigation of ligand binding properties and conformational dynamics of FAT, we have recently initiated structural and biophysical characterization studies on the structurally related domain of Vinculin. Vinculin is a cytoskeletal protein localized to cell-extracellular matrix as well as cell-cell contacts. In both locations, vinculin participates in the linkage of transmembrane receptors, i.e. integrins or cadherins, to the actin cytoskeleton. Vinculin, an essential mammalian gene, functions in the control of cell survival and migration, specifically as a negative regulatory element. Loss of vinculin results in enhanced FAK and paxillin signaling, increased cell migration and survival, and the acquisition of tumorigenic properties in model cell lines. Thus, vinculin exhibits properties of a tumor suppressor. Given its critical biological roles, the regulation of vinculin function is obviously essential. Vinculin is regulated through an intramolecular inhibitory interaction between the N-terminal head domain (Vh) and the C-terminal tail domain (Vt). This interaction obscures docking sites for multiple vinculin-binding partners and several mechanisms have been proposed to relieve this inhibition, including actin, phospholipid (PL) binding and interactions with talin and a-actinin. Although Vt contains binding sites for paxillin and is proposed to undergo structural rearrangement upon interaction with PLs, the sites and consequence of binding these ligands have not been established. Given the importance of these binding interactions in regulating vinculin, several outstanding questions regarding vinculin structure and function are highly significant for physiological regulation of vinculin and control of signaling events downstream of the integrins. NMR, biochemical and biophysical approaches are currently being employed on the vinculin tail domain to investigate actin binding and bundling, PL and paxillin binding interactions as well as studies probing allosteric and dynamic processes. These research efforts are being conducted in collaboration with the Waterman group at NIH and Burridge lab at UNC-CH to integrate molecular analyses with cellular studies.
We have conducted biochemical, biophysical and NMR structural studies of Ig domains contained within the cell adhesion protein palladin. Palladin is a multi-domain, actin-associated protein that is highly conserved among vertebrate species. The Otey laboratory at UNC-CH has shown that palladin exists as multiple isoforms with distinct domain structures, and overexpression of palladin isoforms in cultured cells results in dramatic, isoform-specific alterations in actin organization. Recently, a palladin knockout mouse was generated, and the phenotype was embryonic lethality, demonstrating that palladin plays an essential role in mammalian embryogenesis. To date, however, the precise molecular function of palladin is unknown. In order to understand the role of palladin in actin organization, we have initiated structure/function analyses to test the hypothesis that palladin binds directly to filamentous actin and functions as an actin-crosslinking protein.
Our laboratory employs a multidisciplinary approach to investigate these problems. While our main structural tool is high field NMR spectroscopy, we also employ other biophysical and biochemical methods including various computer modeling and computational approaches, fluorescence spectroscopy, biochemical characterization of binding interactions and enzyme activity. Most of our studies are conducted in collaboration with laboratories that focus on molecular and cellular biological aspects of these problems. This allows us to direct cell-based signaling and transformation analyses.
- P.M. Thompson, C.E. Tolbert, K. Shen, P. Kota, S.M. Palmer, K.M. Plevock, A. Orlova, V.E. Galkin, K. Burridge, E.H. Egelman, N.V. Dokholyan, R. Superfine and S.L. Campbell. Identification of a new actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties. Structure, in press.
- D.C. Brady, M.S. Crowe, M.L. Turski, G.A. Hobbs, X. Yao, A. Chaikuad, S. Knapp, K. Xiao, b.L. Campbell, D. J. Thiele and C.M. Counter.Copper is required for oncogenic BRAF signaling and tumorigenesis. 2014. Nature, in press.
- R. Baker, E.M. Wilkerson, K. Sumita, D.G. Isom, A.T. Sasaki, H.G. Dohlman, S.L. Campbell. Differences in the Regulation of K-Ras and H-Ras Isoforms by monoubiquitination. J Biol Chem. 2013 Dec 27;288(52):36856-62.
- G.A. Hobbs, H.P. Gunawardena and S.L. Campbell. Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases. In Ras Signaling: Methods and Protocols, Humana Press. In press.
- P.M. Thompson, M.R. Beck, S.L. Campbell. Protein-Protein Interaction Analysis by Nuclear Magnetic Resonance Spectroscopy. Protein-Protein Interactions. Methods and Applications 2nd Ed. (Fu H., Meyerkord C., Ed.) Methods in Molecular Biology. In press.
- Hobbs GA, Gunawardena HP, Baker R, S.L. Campbell. Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. Small GTPases. 2013 Sep 12;4(3).
- M.R. Beck, R.D. Dixon, S.M. Goicoechea, G.S. Murphy, J.G. Brungardt, M.T. Beam, P. Srinath, J. Patel, J. Mohiuddin, C.A. Otey, S.L. Campbell. Structure and Function of Palladin’s Actin Binding Domain. Mol Biol. 2013 Sep 23;425(18):3325-37.
- Thievessen, S. Berlemont, K.M. Plevock, S.V. Plotnikov, P.M. Thompson, A. Zemljic-Harpf, R.S. Ross, M.W. Davidson, S.L. Campbell, G. Danuser, et al. Vinculin-actin Interaction Mediates Engagement of Actin Retrograde Flow to Focal Adhesions, but is Dispensable for Actin-dependent Focal Adhesion Maturation. I.J Cell Biol. 2013 Jul 8;202(1):163-77.
- P.M. Thompson, C.E. Tolbert, S.L. Campbell. Vinculin and metavinculin: differences in interactions with F-actin and oligomerization.. 2013 FEBS Lett. 587(8):1220-9.
- C.T. Tolbert, K. Burridge and S.L. Campbell. Vinculin Regulation of F-actin Bundle Formation: What Does it Mean for the Cell? 2013 Cell Adh Migr. Jan 10; 7(2): 219-225.
- G.A. Hobbs, M.G. Bonini, H. P. Gunawardena, X. Chen and S.L. Campbell. Glutathiolated Ras: Characterization and Implications for Ras Activation. 2013 Free Radic Biol Med. Apr;57:221-9.
- R. Baker, S. M. Lewis, E. M. Wilkerson, A. T. Sasaki, L. C. Cantley, B, Kuhlman, H. G. D., S.L. Campbell. *Site-Specific Monoubiquitination Activates Ras by Impeding GTPase Activating Protein Function. 2013. Nat Struct Mol Biol. Jan; 20(1): 46-52.
- Research highlight; Baker, R. Identifying the Mechanism by Which Monoubiquitylation Activates Ras. Nature Rev. Mol. Cell Biol. (2013) in press. NCI press release: www.cancer.gov/newscenter/cancerresearchnews/2012/NewMechanismCancerProgression
- L.Mitchell, A. Hobbs, A. Aghajanian and S.L. Campbell. Redox Regulation of Ras and Rho GTPases: Mechanism and Function, Antioxidants Redox Signaling. 2013 Antioxid Redox Signal. 18(3)250-8. PMID:22657737
- M.F. Davis, L. Zhou, M. Ehrenshaft, K. Ranguelova, H. P. Gunawardena, X. Chen, M.G. Bonini, R. P. Mason, S.L. Campbell. Detection of Ras GTPase protein radicals through immuno-spin trapping. Free Radic Biol Med. 2012 Sep 15;53(6):1339-45. Epub 2012 Jul 20.
- D. Vigil, T.Y. Kim, A. Plachco, A.J. Garton, L. Castaldo, J.A. Pachter, H. Dong, X. Chen, B. Tokar, S.L. Campbell, C.J. Der. ROCK1 and ROCK2 are Required for Non-Small Cell Lung Cancer Anchorage Independent Growth and Invasion. Cancer Res. 2012 Oct 15;72(20):5338-47.
- J. Cable, K. Prutzman, H. Gunawardena, M.D. Schaller, X. Chen and S.L. Campbell. In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase. Biochemistry. 2012 Mar 20;51(11):2213-23. Epub 2012 Mar 8. PMID: 22372511
- L.Mitchell, A.Hobbs, A. Aghajanian and S.L. Campbell. Redox Regulation of Ras and Rho GTPases: Mechanism and Function, Antioxidants Redox Signaling. Antioxid Redox Signal. 2013 Jan 20;18(3):250-8. Epub 2012 Jul 30. Review
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