D Scott Witherow

Duke University Medical Center, Durham, North Carolina, United States

Are you D Scott Witherow?

Claim your profile

Publications (10)41.4 Total impact

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. Here we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. The Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.
    Preview · Article · Feb 2006 · Current Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In addition to their roles in desensitization and signaling of seven-membrane-spanning receptors, beta-arrestins have been more recently implicated in regulating non-seven-membrane-spanning receptor pathways. By using a yeast two-hybrid screen, we identified the inhibitor of NF-kappaB, IkappaBalpha, as a binding partner of beta-arrestin 1. Both beta-arrestin 1 and 2 interact with IkappaBalpha in transfected cells as assessed by immunoprecipitation experiments. Additionally, upstream kinases known to regulate the function of IkappaBalpha, such as IkappaB kinase alpha and beta and NF-kappaB-inducing kinase, were also shown to interact with beta-arrestin. Overexpression of either beta-arrestin 1 or beta-arrestin 2 led to marked inhibition of NF-kappaB activity, as measured by reporter gene activity. Inhibition of NF-kappaB activity was independent of the type of stimulus used for NF-kappaB activation. Conversely, suppression of beta-arrestin 1, but not beta-arrestin 2, expression by using RNA interference led to a 3-fold increase in tumor necrosis factor-stimulated NF-kappaB activity as measured by NF-kappaB mobility-shift analysis. These data uncover a role of beta-arrestins in the regulation of NF-kappaB-mediated gene regulation.
    Preview · Article · Jul 2004 · Proceedings of the National Academy of Sciences
  • D Scott Witherow · Vladlen Z Slepak
    [Show abstract] [Hide abstract]
    ABSTRACT: Regulator of G-protein signaling (RGS) proteins of the R7 subfamily (RGS6, 7, 9, and 11) contain a unique Ggamma-like (GGL) domain that enables their association with the G-protein beta subunit Gbeta5. The existence of these complexes was demonstrated by their purification from native tissues as well as by reconstitution in vitro. According to pulse-chase analysis, Gbeta5 and RGS7 monomers undergo rapid proteolytic degradation in cells, whereas the dimer is stable. Studies of the functional role of Gbeta5-RGS dimers using GTPase activity, ion channel, and calcium mobilization assays showed that, similarly to other RGS proteins, they can negatively regulate G-protein-mediated signal transduction. Protein-protein interactions involving the Gbeta5-RGS7 complex can be studied in cells using fluorescence resonance energy transfer utilizing Gbeta5, RGS, and Galpha subunits fused to the cyan and yellow versions of green fluorescent protein.
    No preview · Article · Feb 2004 · Methods in Enzymology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Gγ-like domain through which they form obligatory dimers with the G protein subunit Gβ5 in vivo. In Caenorhabditis elegans, orthologs of Gβ5·RGS dimers are implicated in regulating both Gαi and Gαq signaling, and in cell-based assays these dimers regulate Gαi/o- and Gαq/11-mediated pathways. However, initial studies with purified Gβ5·RGS6 or Gβ5·RGS7 showed that they only serve as GTPase activating proteins for Gαo. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Gαo or Gαq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7·Gβ5 complex binds to Gαq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gβ5, and Gα subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gβ5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7·Gβ5 complex and cyan fluorescence protein-tagged Gαq, indicating a direct interaction between these molecules.
    Preview · Article · Jul 2003 · Journal of Biological Chemistry
  • D. Scott Witherow · Vladlen Z. Slepak
    [Show abstract] [Hide abstract]
    ABSTRACT: The fifth member of the G protein g the subunit family, G g 5, has been shown to bind exclusively to a subfamily of regulators of G protein signaling (RGS) including RGS6, RGS7, RGS9, and RGS11. This interaction occurs through a G protein gamma-like (GGL) domain present in members of this RGS subfamily and is the only reported instance in which a G g subunit is not bound to a G n subunit. The G g 5-RGS interaction has been demonstrated both in vitro and in vivo and has been shown to stabilize the dimer against proteolytic degradation. GTPase activating protein (GAP) assays suggest that G g 5-RGS7 acts specifically on G f o, however in cell-based assays it also inhibited G f i- and G f q-mediated signaling. The role of the dimer in signaling and the function of G g 5 moiety within the complex are poorly understood. This review summarizes the information about the assembly and function of G g 5-RGS dimers, as well as their posttranslational modifications and localization.
    No preview · Article · May 2003 · Receptors and Channels
  • D Scott Witherow · Vladlen Z Slepak
    [Show abstract] [Hide abstract]
    ABSTRACT: The fifth member of the G protein beta the subunit family, G beta5, has been shown to bind exclusively to a subfamily of regulators of G protein signaling (RGS) including RGS6, RGS7, RGS9, and RGS11. This interaction occurs through a G protein gamma-like (GGL) domain present in members of this RGS subfamily and is the only reported instance in which a G beta subunit is not bound to a G gamma subunit. The G beta5-RGS interaction has been demonstrated both in vitro and in vivo and has been shown to stabilize the dimer against proteolytic degradation. GTPase activating protein (GAP) assays suggest that G beta5-RGS7 acts specifically on G alphao, however in cell-based assays it also inhibited G alphai- and G alphaq-mediated signaling. The role of the dimer in signaling and the function of G beta5 moiety within the complex are poorly understood. This review summarizes the information about the assembly and function of G beta5-RGS dimers, as well as their posttranslational modifications and localization.
    No preview · Article · Feb 2003 · Receptors and Channels
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: RGS proteins regulate G protein-mediated signalling pathways through direct interaction with the Galpha subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein beta subunit Gbeta5 via a characteristic Ggamma-like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7-Gbeta5 complex in inflammatory cells, we performed immunocytochemistry, RT-PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non-stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF-alpha In addition, RGS7-positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7-Gbeta5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post-traumatic inflammatory responses in the central nervous system.
    Full-text · Article · Mar 2002 · European Journal of Neuroscience
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Regulators of G protein signaling RGS4 and RGS7 accelerate the kinetics of K(+) channels (GIRKs) in the Xenopus oocyte system. Here, via quantitative analysis of RGS expression, we reveal biphasic effects of RGSs on GIRK regulation. At low concentrations, RGS4 inhibited basal GIRK activity, but stimulated it at high concentrations. RGS7, which is associated with the G protein subunit G beta 5, is regulated by G beta 5 by two distinct mechanisms. First, G beta 5 augments RGS7 activity, and second, it increases its expression. These dual effects resolve previous controversies regarding RGS4 and RGS7 function and indicate that they modulate signaling by mechanisms supplementary to their GTPase-activating protein activity.
    Full-text · Article · Apr 2001 · FEBS Letters
  • Source
    D S Witherow · Q Wang · K Levay · J L Cabrera · J Chen · G B Willars · V Z Slepak
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Gα subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Gγ -like domain, also has the unique ability to interact with the G protein β subunit Gβ5. Here, we examined the behavior of Gβ5, RGS7, RGS9, and Gα in tissue extracts using immunoprecipitation and conventional chromatography. Native Gβ5 and RGS7 from brain, as well as photoreceptor-specific Gβ5L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gβ5 nor Gβ5-free RGS could be detected. Co-expression in COS-7 cells of Gβ5 dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gβ5:RGS stoichiometry in a manner similar to Gβγ complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gβ5-RGS and their coupled expression argue that in vivo, Gβ5and Gγ-like domain-containing RGSs only exist as heterodimers. Native Gβ5-RGS7 did not co-precipitate or co-purify with Gαo or Gαq; nor did Gβ5 L-RGS9 with Gαt. However, in transfected cells, RGS7 and Gβ5-RGS7 inhibited Gαq-mediated Ca2+ response to muscarinic M3 receptor activation. Thus, Gβ5-RGS dimers differ from other RGS proteins in that they do not bind to Gα with high affinity, but they can still inhibit G protein signaling.
    Full-text · Article · Sep 2000 · Journal of Biological Chemistry
  • [Show abstract] [Hide abstract]
    ABSTRACT: A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Gα subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Gγ -like domain, also has the unique ability to interact with the G protein β subunit Gβ5. Here, we examined the behavior of Gβ5, RGS7, RGS9, and Gα in tissue extracts using immunoprecipitation and conventional chromatography. Native Gβ5 and RGS7 from brain, as well as photoreceptor-specific Gβ5L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gβ5 nor Gβ5-free RGS could be detected. Co-expression in COS-7 cells of Gβ5 dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gβ5:RGS stoichiometry in a manner similar to Gβγ complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gβ5-RGS and their coupled expression argue that in vivo, Gβ5and Gγ-like domain-containing RGSs only exist as heterodimers. Native Gβ5-RGS7 did not co-precipitate or co-purify with Gαo or Gαq; nor did Gβ5 L-RGS9 with Gαt. However, in transfected cells, RGS7 and Gβ5-RGS7 inhibited Gαq-mediated Ca2+ response to muscarinic M3 receptor activation. Thus, Gβ5-RGS dimers differ from other RGS proteins in that they do not bind to Gα with high affinity, but they can still inhibit G protein signaling.
    No preview · Article · Aug 2000 · Journal of Biological Chemistry

Publication Stats

477 Citations
41.40 Total Impact Points

Institutions

  • 2004-2006
    • Duke University Medical Center
      • • Department of Pharmacology and Cancer Biology
      • • Department of Medicine
      Durham, North Carolina, United States
    • Duke University
      Durham, North Carolina, United States
  • 2000-2003
    • University of Miami Miller School of Medicine
      • Department of Molecular and Cellular Pharmacology
      Miami, FL, United States