Sharon Campbell

Research: Regulator molecules in cell growth control and cell adhesion

Sharon Campbell

Professor of Biochemistry and Biophysics
(PhD - Yale University)

120 Mason Farm Road, CB# 7260
3093 Genetic Medicine
Chapel Hill, NC 27599-7260

Campbell Lab Website


  • Battle Distinguished Cancer Research Award - 2014
  • 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.

Three RAS genes (HRAS, KRAS and NRAS) comprise the most frequently mutated oncogene family in cancer, with single point mutations predominately (99%) localized to codons 12, 13 and 61. A common feature of these point mutations is that they render Ras insensitive to down regulation by cellular factors called GTPase activating proteins (GAPs) that catalyze hydrolysis of GTP, resulting in constitutive, oncogenic signaling.  As such, they have historically been considered oncogenic equivalents. However, recent observations suggest that amino acid and codon-specific Ras proteins show differences in their biochemical properties, ability to engage effectors, signaling and tumorigenic properties.  Differences have also been observed in the response and resistance to specific anti-cancer therapies. These differences are likely to have important clinical and biological implications.  Our lab is characterizing the molecular properties of amino acid-, codon- and isoform-specific Ras mutant proteins to better understand how these differences modulate Ras tumorigenicity.

Another research focus of the lab lies in elucidating novel mechanisms by which post-translational modifications within the core Ras guanine nucleotide binding domain modulate its 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 monoubiquitylation of Ras leads to hyper-activation. Mutation of the primary site of monoubiquitylation 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 ubiquitylated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitylation 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).  In addition, we have initiated studies to determine whether other lysine-specific post-translational modifications modulate Ras function.

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 Ras and Rho GTPase post-translational modifications, including monoubiquitylation 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 Metavinculin

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, metavinculin, paxillin 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, FAT domain conformational dynamics in FAK function and characterizing FAK inhibitors that bind to the FAT domain.

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 actin and is proposed to undergo structural rearrangement upon interaction with PLs, the sites and consequence of binding these ligands have not been well characterized. 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 binding interactions as well as studies probing allosteric and dynamic processes.  These research efforts are being conducted in collaboration with the Waterman group and Alushin groups at NIH and Burridge lab at UNC-CH to integrate molecular analyses with cellular studies.

Research Tools

Our laboratory employs multidisciplinary approaches 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 adhesion, motility, signaling and transformation analyses.

RECENT PUBLICATIONS  pubmed.png(click for full publication list

  • Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions. Case LB, Baird MA, Shtengel G, Campbell SL, Hess HF, Davidson MW, Waterman CM. Nat Cell Biol. 2015 Jul;17(7):880-92. doi: 10.1038/ncb3180. Epub 2015 Jun 8.
  • Protein-protein interaction analysis by nuclear magnetic resonance spectroscopy. Thompson PM, Beck MR, Campbell SL. Methods Mol Biol. 2015;1278:267-79. doi: 10.1007/978-1-4939-2425-7_16.
  • Rac1 modification by an electrophilic 15-deoxy Δ(12,14)-prostaglandin J2 analog. Wall SB, Oh JY, Mitchell L, Laube AH, Campbell SL, Renfrow MB, Landar A. Redox Biol. 2015;4:346-54. doi: 10.1016/j.redox.2015.01.016. Epub 2015 Feb 3.
  • Redox regulation of Rac1 by thiol oxidation. Hobbs GA, Mitchell LE, Arrington ME, Gunawardena HP, DeCristo MJ, Loeser RF, Chen X, Cox AD,Campbell SL. Free Radic Biol Med. 2015 Feb;79:237-50. doi: 10.1016/j.freeradbiomed.2014.09.027. Epub 2014 Oct 5.
  • Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma. Burd CE, Liu W, Huynh MV, Waqas MA, Gillahan JE, Clark KS, Fu K, Martin BL, Jeck WR, Souroullas GP, Darr DB, Zedek DC, Miley MJ, Baguley BC, Campbell SL, Sharpless NE. Cancer Discov. 2014 Dec;4(12):1418-29. doi: 10.1158/2159-8290.CD-14-0729. Epub 2014 Sep 24
  • Identification of an actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties. Thompson PM, Tolbert CE, Shen K, Kota P, Palmer SM, Plevock KM, Orlova A, Galkin VE, Burridge K, Egelman EH, Dokholyan NV, Superfine R, Campbell SL. Structure. 2014 May 6;22(5):697-706. doi: 10.1016/j.str.2014.03.002. Epub 2014 Mar 27.
  • Biophysical and proteomic characterization strategies for cysteine modifications in Ras GTPases. Hobbs GA, Gunawardena HP, Campbell SL. Methods Mol Biol. 2014;1120:75-96. doi: 10.1007/978-1-62703-791-4_6. Erratum in: Methods Mol Biol. 2014;1120:e1.
  • Phosphorylation at Y1065 in Vinculin Mediates Actin Bundling, Cell Spreading, and Mechanical Responses to Force C.E. Tolbert , P.M. Thompson ,  R. Superfine, K. Burridge , S.L. Campbell  . Biochemistry. 2014 Aug 12.
  • Rho GTPases, oxidation, and cell redox control. G.A. Hobbs, B. Zhou, A.D. Cox, S.L. Campbell   Small GTPases. 2014;5:e28579.
  • Identification of a new actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties. 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.  Structure 2014 May 6;22 (5):697-706.
  • Copper is required for oncogenic BRAF signaling and tumorigenesis.  D.C. Brady,  M.S. Crowe, M.L. Turski, G.A. Hobbs, X. Yao, A. Chaikuad, S. Knapp, K. Xiao, S.L. Campbell, D. J. Thiele and C.M. Counter. Nature 2014 May 22;509(7501):492-6.
  • Biophysical and proteomic characterization strategies for cysteine modifications in Ras GTPases. G.A. Hobbs, H.P. Gunawardena, S.L. Campbell  Methods Mol Biol. 2014;1120:75-96.
  • Differences in the Regulation of K-Ras and H-Ras Isoforms by monoubiquitination.  R. Baker, E.M. Wilkerson, K. Sumita, D.G. Isom, A.T. Sasaki, H.G. Dohlman, S.L. Campbell.  J Biol Chem. 2013 Dec 27;288 (52):36856-62.
  • Protein-Protein Interactions. P.M. Thompson, M.R. Beck, S.L. Campbell. Protein-Protein Interaction Analysis by Nuclear Magnetic  Resonance Spectroscopy.  Methods and Applications 2nd Ed. (Fu H., Meyerkord C., Ed.) Methods in Molecular Biology. In press.
  • Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. G.A. Hobbs, H.P. Gunawardena, R. Baker, S.L. Campbell. Small GTPases. 2013 Sep 12;4(3).
  • Structure and Function of Palladin’s Actin Binding Domain. 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. Campbel   Mol Biol.  2013 Sep 23;425(18):3325-37
  • Commentary: Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function. G.A. Hobbs, R. Baker and S.L. Campbell. Small GTPases, 2013 Sep 12;4(3).
  • Vinculin-actin Interaction Mediates Engagement of Actin Retrograde Flow to Focal Adhesions, but is Dispensable for Actin-dependent Focal Adhesion Maturation. I. 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., 2013 J Cell Biol., Jul;202(1):163-77.
  • Vinculin and metavinculin: differences in interactions with F-actin and oligomerization. P.M. Thompson, C.E. Tolbert, S.L. Campbell. 2013 FEBS Lett. 587(8):1220-9.
  • Vinculin Regulation of F-actin Bundle Formation: What Does it Mean for the Cell? C.T. Tolbert, K. Burridge and S.L. Campbell. 2013 Cell Adh Migr. Jan 10; 7(2): 219-225.
  • Glutathiolated Ras: Characterization and Implications for Ras Activation. G.A. Hobbs, M.G. Bonini, H. P. Gunawardena, X. Chen and S.L. Campbell. 2013 Free Radic Biol Med. Apr;57:221-9.
  • *Site-Specific Monoubiquitination Activates Ras by Impeding GTPase Activating Protein Function. R. Baker, S. M. Lewis, E. M. Wilkerson, A. T. Sasaki, L. C. Cantley, B, Kuhlman, H. G. D., S.L. Campbell. 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. NCI press release:
  • Redox Regulation of Ras and Rho GTPases: Mechanism and Function, L.Mitchell, A. Hobbs, A. Aghajanian and S.L. Campbell, Antioxidants Redox Signaling. 2013 Antioxid Redox Signal. 18(3)250-8. PMID:22657737

Lab Contact:

Lab Room: 3100A-B Genetic Medicine
Lab Phone: (919) 966-6781



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