Last Updated: 7/25/2006

Research Interests
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 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.

Research Tools
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.

Recent Accomplishments and Honors
Hettleman award - 2001
Jefferson-Pilot award - 1998

Publications
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.

J. Heo and SL Campbell. Nitric Oxide and Cell Signaling. Nitric Oxide, Cell Signaling and Gene Expression. Marcel Dekker, Inc. (2005).

J. Heo and SL Campbell. Mechanism of redox-mediated guanine nucleotide exchange on redox-active Rho GTPases. J Biol Chem. 2005 Sep 2;280(35):31003-10.

J Heo, SL Campbell. Mechanism of redox-mediated guanine nucleotide exchange on redox-active Rho gtpases, J Biol Chem 2005 Jun 30.

J Heo, R Thapar, SL Campbell. Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors, Biochemistry, 2005 May 3;44(17):6573-85.

J. Heo and SL Campbell. Superoxide Anion Radical Modulates the Activity of Ras and Ras related GTPases by a Radical-based Mechanism Similar to That of Nitric Oxide. J Biol Chem. 2005 Apr 1;280(13):12438-45.

J. Heo, KC Prutzman, V Mocanu and SL Campbell. Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation. J Mol Biol. 2005 Mar 11;346(5):1423-40.

J Heo, SL Campbell. Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide, J Biol Chem. 2005 Apr 1;280(13):12438-45. Epub 2005 Jan 31.

M Hekman, A Fischer, LP Wennogle, YK Wang, SL Campbell, UR Rapp. Novel C-Raf phosphorylation sites: serine 296 and 301 participate in Raf regulation. FEBS Lett. 2005 Jan 17;579(2):464-8.

J Heo, KC Prutzman, V Mocanu, SL Campbell. Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation, J Mol Biol. 2005 Mar 11;346(5):1423-40. Epub 2005 Jan 12.

SG Chaney, SL Campbell, B Temple, E Bassett, Y Wu and M Faldu. Recognition and Processing of Platinum-DNA Adducts. Crit Rev Oncol Hematol. 2005 Jan;53(1):3-11.

RD Dixon, Y Chen, F Ding, SD Khare, KC Prutzman, MD Schaller, SL Campbell, NV Dokholyan. New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate. Structure (Camb). 2004 Dec;12(12):2161-71.

R Thapar, JG Williams, SL Campbell. NMR characterization of full-length farnesylated and non-farnesylated H-Ras and its implications for Raf activation. J Mol Biol. 2004 Nov 5;343(5):1391-408.

Y Wu, P Pradhan, J Havener, G Boysen, JA Swenberg, SL Campbell, SG Chaney. NMR solution structure of an oxaliplatin 1,2-d(GG) intrastrand cross-link in a DNA dodecamer duplex. J Mol Biol. 2004 Aug 27;341(5):1251-69.

J Heo, G Gao, SL Campbell. pH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange. Biochemistry. 2004 Aug 10;43(31):10102-11.

PA Solski, RS Wilder, KL Rossman, J Sondek, AD Cox, SL Campbell, CJ Der. Requirement for C-terminal sequences in regulation of Ect2 guanine nucleotide exchange specificity and transformation. J Biol Chem. 2004 Jun 11;279(24):25226-33. Epub 2004 Apr 8.

KC Prutzman, G Gao, ML King, VV Iyer, GA Mueller, MD Schaller, SL Campbell. The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation. Structure (Camb). 2004 May;12(5):881-91.

G Gao, JG Williams, SL Campbell. Protein-protein interaction analysis by nuclear magnetic resonance spectroscopy. Methods Mol Biol. 2004;261:79-92. Review.

AE Karnoub, M Symons, SL Campbell, CJ Der. Molecular basis for Rho GTPase signaling specificity. Breast Cancer Res Treat. 2004 Mar;84(1):61-71.

J Heo, SL Campbell. Mechanism of p21Ras S-nitrosylation and kinetics of nitric oxide-mediated guanine nucleotide exchange. Biochemistry. 2004 Mar 2;43(8):2314-22.

G Gao, KC Prutzman, ML King, DM Scheswohl, EF DeRose, RE London, MD Schaller, SL Campbell. NMR solution structure of the focal adhesion targeting domain of focal adhesion kinase in complex with a paxillin LD peptide: evidence for a two-site binding model. J Biol Chem. 2004 Feb 27;279(9):8441-51. Epub 2003 Dec 7.

E-mail: campbesl@med.unc.edu
Telephone: (919) 966-7139
FAX: (919) 966-2852
Address: 530A Mary Ellen Jones Bldg. Chapel Hill, NC 27599-7260
URL: http://www.med.unc.edu/biochem/scampbell/

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