Professor of Biochemistry and Biophysics, UNC-CH
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 the mechanism by which nitric oxide and superoxide anion radical mediate guanine nucleotide dissociation of select Ras and Rho GTPases. Current research projects in the Campbell laboratory include: redox regulation of Ras superfamily GTPases using kinetic and spectroscopic approaches; structural, biophysical and biochemical studies of wild type and variant Ras and Rho family GTPase proteins, as well as the identification, characterization and structural elucidation of factors that act on Ras 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. In collaboration with the Schaller laboratory (UNC-CH), we have initiated NMR structural investigations on the cell adhesion proteins, Focal Adhesion Kinase (FAK) and Vinculin.
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 have recently 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 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 PL binding in regulating vinculin and the hypothesis that paxillin is a key binding partner in the regulation of biological functions of vinculin, these outstanding questions regarding vinculin structure and function are highly significant for the physiological regulation of vinculin and control of signaling events downstream of the integrins. NMR approaches, biochemical and biophysical approaches are being employed to investigate Vt self-association, PL and paxillin binding interactions and probe conformational dynamics and dynamic processes associated with Vinculin function.
We have recently initiated biochemical, biophysical and NMR structural studies of Ig domains contained within the cell adhesion protein palladin in collaboration with the Otey laboratory in the department of Cell and Molecular Physiology at UNC-CH. Palladin is a multi-domain, actin-associated protein that is highly conserved among vertebrate species. The Otey laboratory 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.
Platinated DNA Adducts
Platinum anticancer agents are widely used in cancer chemotherapy. These platinum complexes appear to kill dividing cells by forming platinum-DNA adducts which interfere with DNA replication and cell division. Recent research has suggested that platinum complexes with the diaminocyclohexane carrier ligand (oxaliplatin) may offer therapeutic advantages because they have reduced toxicity and are often effective in cancer cell lines with resistance to currently used platinum complexes. We have recently solved the NMR solution structure of an oxaliplatin-DNA adduct by NMR, in collaboration with the Chaney laboratory at UNC-CH. NMR structural studies are currently in progress to compare directly, in the same sequence context, structural and dynamic differences between oxaliplatin and cis-platinated DNA adducts alone and in complex with DNA binding proteins that discriminate between these distinct platinated adducts in vivo.
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.
A. Aghajanian., E.S. Wittchen, S.L. Campbell and K Burridge. Direct activation of RhoA by reactive oxygen species requires a redox-sensitive motif. PLoS ONE, 2009 4(11): e8045.
- S.M. Palmer, M.P. Playford, S.W. Craig, M.D. Schaller, S.L. Campbell. Lipid binding to the tail domain of vinculin: specificity and the role of the N and C termini. J Biol Chem. 2009 Mar 13;284(11):7223-31.
- S.M. Palmer, M.D. Schaller, S.L. Campbell. Vinculin tail conformation and self-association is independent of pH and H906 protonation. Biochemistry. 2008 Nov 25;47(47):12467-75.
- S.M. Palmer, S.L. Campbell. Backbone 1H, 13C and 15N NMR Assignments of the tail domain of vinculin. Biomolecular NMR Assignments. 2008 June;2:69-71.
- R.D. Dixon, S.L. Campbell. 1H, 15N, and 13C NMR chemical shift assignments for the lg3 domain of palladin. Biomoelcular NMR Assignments. 2008 June;2:51-53.
- R.D. Dixon, D.K. Arneman, A.S. Rachlin, N.R. Sundaresan, M.J. Costello, *S.L.Campbell, *C.A. Otey CA. Palladin is an actin cross-linking protein that uses immunoglobulin-like domains to bind filamentous actin. J Biol Chem. 2008 Mar 7;283(10):6222-31. *Equal Contribution
- K.W. Raines, M.G. Bonini, S.L.Campbell. Nitric Oxide Cell Signaling: S-nitrosation of Ras Superfamily GTPases. Cardiovascular Research. 2007 July 15;75(2):229-39
- Y. Wu, D. Bhattacharyya, C.L. King, I. Baskerville-Abraham, S.H. Huh, G. Boysen, J.A. Swenberg, B. Temple, S.L.Campbell*, S.G. Chaney*. Solution structures of a DNA dodecamer duplex with and without a cisplatin 1,2-d(GG) cross-link: comparison with the same DNA duplex containing an oxaliplatin1,2-d(GG) intrastrand cross-link. Biochemistry. 2007 Jun 5;46(22):6477-87. *Equal Contribution
- J. Heo, K.W. Raines, V. Mocanu, S.L. Campbell. Redox regulation of RhoA. Biochemistry. 2006 Dec 5;45(48):14481-9.
- J. Heo and SL Campbell. Ras regulation by reactive oxygen and nitrogen species. Biochemistry. 2006 Feb 21;45(7):2200-10.
- F. Ding, K.C Prutzman, SL Campbell, N.V. Dokholyan. Topological determinants of protein domain swapping. Structure. 2006 Jan;14(1):5-14.
120 Mason Farm Rd,
Campus Box # 7260
3093 Genetic Medicine
Chapel Hill, NC 27599
Lab Location: 3100 A-C Genetic Med