Associate Professor
Ph.D., Syracuse University, 1988
The long term goal of my research is to define new gene products involved in cytokine-mediated cellular changes and to determine their role in these events. Interferon-γ was the cytokine used for the early studies. In addition to imparting resistance to viral infection, for which they were originally identified, IFNs influence the growth and differentiation of a wide variety of cell types. IFN-γ also has profound immune modulatory effects. Upon exposure to IFN-γ, cells undergo a multitude of changes, some of these include alterations in cell growth, changes in cell motility, induction of MHC expression, cytotoxicity, and secretion of a number of cytokines. Toward the goal of identifying cytokine-regulated genes, differential display RT-PCR was used to identify IFN-γ-regulated genes in murine bone marrow-derived macrophages. I have concentrated on a novel protein that I identified from this screen, a new member of the family of guanylate binding proteins (GBPs), named murine GBP-2 (mGBP-2) (Vestal et al. 1998). GBPs are a closely related family of 65-67 kDa IFN-induced GTPases.
Structural properties of GBPs. GBPs share two important structural features with the Ras superfamily of GTPases. The first is the presence of a guanine nucleotide binding pocket. Within the first of the conserved regions of the GTP binding pocket, GBPs have amino acids identical to those of the Ras superfamily and dynamin, to which GBPs are distantly related. Point mutations at two of these positions in both members of the Ras family and dynamin result in dominant negative proteins that have been used to define the involvement of these proteins within their respective pathways and to determine the role of regulators and effectors in these processes. The corresponding point mutation to Ras (S17N) has been generated in mGBP-2 (S52N) and the resultant protein shows reduced binding to guanine nucleotides.
The second feature found in members of the Ras superfamily and shared between most members of the GBP family, including mGBP-2, is a site for isoprenoid modification, called a CaaX sequence (a cysteine residue (C) followed by two (usually) aliphatic amino acids (a) and another amino acid (X)). Isoprenylation is a multistep process that begins with the attachment of either a C-15 farnesyl or C-20 geranylgeranyl isoprenoid to the cysteine residue (C) of the terminal CaaX moiety. We have demonstrated that 3 of the mammalian GBPs are indeed prenylated in vivo (Nantais et al., 1996; Vestal et al., 1996; Vestal et al., 1998). We have also demonstrated that isoprenylation is required for the targeting of mGBP-2 to intracellular membranes (Vestal et al., 2000).
Intracellular location of mGBP-2. Recently we have shown that mGBP-2 is found within IFN-treated cells in both a granular distribution throughout their cytoplasm and in a heterogeneous population of intracellular vesicles (Vestal et al., 2000). Targeting of mGBP-2 to these intracellular vesicles requires the addition of the geranylgeranyl isoprenoid but does not require other IFN-responsive proteins, based on the observation that vesicular localization occurs in cells unable to respond to IFNs.
mGBP-2-mediated changes in cell growth. Stable cell lines that express mGBP-2 and the GTP binding mutant at several different steady state levels of protein have been generated in NIH 3T3 cells. Somewhat surprisingly, mGBP-2-expressing cells grow at an accelerated rate compared to control transfectants. IFN-γ treatment of untransfected NIH 3T3 cells also accelerated growth. The GTP binding mutant of mGBP-2 does not grow abnormally, indicating that wild type GTPase activity is required for these growth alterations (Gorbacheva et al., 2002).
mGBP-2-mediated changes in actin cytoskeleton, adhesion, and migration. IFN-γ treatment of NIH 3T3 cells results in reduced spreading on fibronectin. Cells expressing mGBP-2 show the same reduction in the ability to spread on fibronectin, demonstrating that mGBP-2 is sufficient to mediate these changes in the absence of other IFN-induced proteins. In addition, the S52N GTP binding mutant is able to block IFN-γ mediated reduction in cell spreading, indicating that mGBP-2 is also necessary for this phenotype. In addition we have shown that mGBP-2 alters the actin cytoskeleton of cells constitutively expressing the protein. The finding that mGBP-2 alters cytoskeleton, cell attachment and migration is an exciting observation because this is one area of IFN responses that is very poorly understood. A better understanding of how mGBP-2 facilitates these changes has major implications for a variety of processes, not the least of which are cell migration and recruitment of immune cells into inflammatory sites.
Olszewski, M. A., Gray, J., and Vestal, D.J. (2006). In silico genomic analysis of the human and murine guanylate binding protein (GBP) gene clusters. J. Interferon Cytokine Res. 26: 328-352.
Balasubramian, S., Nada, S., and Vestal, D.J. (2006). The interferon-induced GTPase, mGBP-2, confers resistance to paclitaxel-induced cytotoxicity without inhibiting multinucleation. Cellular and Molecular Biology 52: 43-49.
Vestal, D.J. (2005). The guanylate binding proteins (GBPs): Pro-inflammatory cytokine-induced members of the dynamin superfamily with unique GTPase activity. J. Interferon Cytokine Res. 25: 435-443.
Carter, C., Gorbacheva, V.Y., and Vestal, D.J. (2005). Inhibition of VSV and EMCV replication by the interferon-induced GTPase, mGBP-2: Differential requirement for wild-type GTP binding domain. Archives of Virology, 150: 1213-1220.
Gorbacheva, V.Y., Lindner, D., Sen, G.C., and Vestal, D.J. (2002). The interferon (IFN)-induced GTPase, mGBP-2: Role in IFN-y-induced murine fibroblast proliferation. J. Biol. Chem. 277: 6080-6087.
Vestal, D.J., Gorbacheva, V.Y., and Sen, G.C. (2000). Different subcellular localizations for the related interferon-induced GTPases, mGBP-1 and mGBP-2: Implications for different functions? J. Interferon Cytokine. Res. 20: 991-1000.
Patel, R., Vestal, D.J., Xu, Z., Bandyopadhyay,S., Guo, W., Erme, S.,Williams, B.R.G., and Sen, G.C. (1999). DRBP 76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR. J. Biol. Chem. 274: 20432-20437.
Vestal, D.J., Buss, J.E., McKercher, S.R., Jenkins, N.A., Copeland, K.G., Kelner, G. S., Asundi, V.K. and Maki, R.A. (1998). Murine GBP-2: A new IFN-γ-induced member of the GBP family of GTPases from macrophages. J. Interferon and Cytokine Res. 18: 977-985.
McKercher, S.R., Torbett, B.E., Anderson, K.L., Henkel, G., Vestal, D.J., Baribault, H., Klemsz, M., Feeney, A.J., Wu, G.E., Paige, C.J., and Maki, R.A. (1996). Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15: 5647-5658.
Nantais, D.E., Schwemmle, M., Stickney, J.T., Vestal, D.J., and Buss, J.E. (1996). Prenylation of an Interferon-γ-induced GTP-binding Protein: the human guanylate binding protein, huGBP-1. J. Leuk. Biol. 60: 423-431.
Vestal, D.J., Buss, J.E., Kelner, G.S., Maciejewski, D., Asundi, V.K., and Maki, R.A. (1996). Rat p67 GBP is induced by interferon-γ and isoprenoid-modified in macrophages. Biochem. Biophys. Res. Commun. 224: 528-534.