G M Bokoch

The Scripps Research Institute, La Jolla, CA, United States

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Publications (227)1626.15 Total impact

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    ABSTRACT: Spatial control of RhoGTPase-inactivating GAP components remains largely enig-matic. We describe a brain-specific RhoGAP splice variant, BARGIN (BGIN), which comprises a combination of BAR, GAP, and partial CIN phosphatase domains spliced from adjacent SH3BP1 and CIN gene loci. Excision of BGIN exon 2 results in recoding of a 42–amino acid N-terminal stretch. The partial CIN domain is a poly-ubiquitin (poly-Ub)–binding module that facilitates BGIN distribution to membranous and detergent-insoluble fractions. Poly-Ub/BGIN interactions support BGIN-mediated inactivation of a membranous Rac1 population, which consequently inactivates membrane-localized Rac1 effector systems such as reactive oxygen species (ROS) generation by the Nox1 complex. Given that Ub aggregate pathology and proteotoxicity are central themes in various neurodegenerative disorders, we investigated whether BGIN/Rac1 signaling could be involved in neurodegenerative proteotoxicity. BGIN/ Ub interactions are observed through colocalization in tangle aggregates in the Alzheimer's disease (AD) brain. Moreover, enhanced BGIN membrane distribution correlates with reduced Rac1 activity in AD brain tissue. Finally, BGIN contributes to Rac1 inhibition and ROS genera-tion in an amyloid precursor protein (APP) proteotoxicity model. These results suggest that BGIN/poly-Ub interactions enhance BGIN membrane distribution and relay poly-Ub signals to enact Rac1 inactivation, and attenuation of Rac1 signaling is partially dependent on BGIN in a proteotoxic APP context. INTRODUCTION
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    [Show abstract] [Hide abstract]
    ABSTRACT: Spatial control of RhoGTPase-inactivating GAP components remains largely enig-matic. We describe a brain-specific RhoGAP splice variant, BARGIN (BGIN), which comprises a combination of BAR, GAP, and partial CIN phosphatase domains spliced from adjacent SH3BP1 and CIN gene loci. Excision of BGIN exon 2 results in recoding of a 42–amino acid N-terminal stretch. The partial CIN domain is a poly-ubiquitin (poly-Ub)–binding module that facilitates BGIN distribution to membranous and detergent-insoluble fractions. Poly-Ub/BGIN interactions support BGIN-mediated inactivation of a membranous Rac1 population, which consequently inactivates membrane-localized Rac1 effector systems such as reactive oxygen species (ROS) generation by the Nox1 complex. Given that Ub aggregate pathology and proteotoxicity are central themes in various neurodegenerative disorders, we investigated whether BGIN/Rac1 signaling could be involved in neurodegenerative proteotoxicity. BGIN/ Ub interactions are observed through colocalization in tangle aggregates in the Alzheimer's disease (AD) brain. Moreover, enhanced BGIN membrane distribution correlates with reduced Rac1 activity in AD brain tissue. Finally, BGIN contributes to Rac1 inhibition and ROS genera-tion in an amyloid precursor protein (APP) proteotoxicity model. These results suggest that BGIN/poly-Ub interactions enhance BGIN membrane distribution and relay poly-Ub signals to enact Rac1 inactivation, and attenuation of Rac1 signaling is partially dependent on BGIN in a proteotoxic APP context. INTRODUCTION
    Molecular Biology of the Cell. 01/2013;
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    [Show abstract] [Hide abstract]
    ABSTRACT: Spatial control of RhoGTPase-inactivating GAP components remains largely enigmatic. We describe here a brain-specific RhoGAP splice variant, BARGIN (BGIN) which comprises a combination of BAR, GAP and partial CIN phosphatase domains spliced from adjacent SH3BP1 and CIN gene loci. Excision of BGIN exon 2 results in recoding of a 42aa N-terminal stretch. The partial CIN domain is a poly-Ubiquitin (poly-Ub) binding module that facilitates BGIN distribution to membranous and detergent-insoluble fractions. Poly-Ub/BGIN interactions support BGIN-mediated inactivation of a membranous Rac1 population, which consequently inactivates membrane-localized Rac1 effector systems such as ROS generation by the Nox1 complex. Since Ub aggregate pathology and proteotoxicity are central themes in various neurodegenerative disorders, we investigated whether BGIN/Rac1 signaling could be involved in neurodegenerative proteotoxicity. BGIN/Ub interactions are observed through colocalization in tangle aggregates in Alzheimer's (AD) brain. Moreover, enhanced BGIN membrane distribution and correlates with reduced Rac1 activity in AD brain tissue. Lastly, BGIN contributes to Rac1 inhibition and ROS generation in an APP proteotoxicity model. These results suggest that BGIN/poly-Ub interactions enhance BGIN membrane distribution and relays poly-Ub signals to enact Rac1 inactivation, and attenuation of Rac1 signaling is partially dependent on BGIN in a proteotoxic APP context.
    Molecular biology of the cell 12/2012; · 5.98 Impact Factor
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    ABSTRACT: The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.
    Developmental Cell 08/2012; 23(2):397-411. · 10.37 Impact Factor
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    ABSTRACT: Cell motility requires the spatial and temporal coordination of forces in the actomyosin cytoskeleton with extracellular adhesion. The biochemical mechanism that coordinates filamentous actin (F-actin) assembly, myosin contractility, adhesion dynamics, and motility to maintain the balance between adhesion and contraction remains unknown. In this paper, we show that p21-activated kinases (Paks), downstream effectors of the small guanosine triphosphatases Rac and Cdc42, biochemically couple leading-edge actin dynamics to focal adhesion (FA) dynamics. Quantitative live cell microscopy assays revealed that the inhibition of Paks abolished F-actin flow in the lamella, displaced myosin IIA from the cell edge, and decreased FA turnover. We show that, by controlling the dynamics of these three systems, Paks regulate the protrusive activity and migration of epithelial cells. Furthermore, we found that expressing Pak1 was sufficient to overcome the inhibitory effects of excess adhesion strength on cell motility. These findings establish Paks as critical molecules coordinating cytoskeletal systems for efficient cell migration.
    The Journal of Cell Biology 06/2011; 193(7):1289-303. · 9.69 Impact Factor
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    ABSTRACT: NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been implicated in several physiological and pathophysiological processes. To date seven members of this family have been reported, including Nox1-5 and Duox1 and 2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and requires two cytosolic regulators, the organizer subunit NoxO1 and the activator subunit NoxA1, as well as the binding of Rac1 GTPase, for its activity. Recently, we identified the c-Src substrate proteins Tks4 and Tks5 as functional members of a p47(phox)-related organizer superfamily. As a functional consequence of this interaction, Nox1 localizes to invadopodia, actin-rich membrane protrusions of cancer cells which facilitate pericellular proteolysis and invasive behavior. Here, we report that Tks4 and Tks5 directly bind to NoxA1. Moreover, the integrity of the N-terminal PRR of NoxA1 is essential for this direct interaction with the Tks proteins. When the PRR in NoxA1 is disrupted, Tks proteins cannot bind NoxA1 and lose their ability to support Nox1-dependent ROS generation. Consistent with this, Tks4 and Tks5 are unable to act as organizers for Nox2 because of their inability to interact with p67(phox), which lacks the N-terminal PRR, thus conferring a unique specificity to Tks4 and 5. Taken together, these results clarify the molecular basis for the interaction between NoxA1 and the Tks proteins and may provide new insights into the pharmacological design of a more effective anti-metastatic strategy.
    European journal of cell biology 02/2011; 90(2-3):164-71. · 3.31 Impact Factor
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    ABSTRACT: The NADPH oxidase (Nox) proteins catalyze the regulated formation of reactive oxygen species (ROS), which play key roles as signaling molecules in several physiological and pathophysiological processes. ROS generation by the Nox1 member of the Nox family is necessary for the formation of extracellular matrix (ECM)-degrading, actin-rich cellular structures known as invadopodia. Selective inhibition of Nox isoforms can provide reversible, mechanistic insights into these cellular processes in contrast to scavenging or inhibition of ROS production. Currently no specific Nox inhibitors have been described. Here, by high-throughput screening, we identify a subset of phenothiazines, 2-acetylphenothiazine (here referred to as ML171) (and its related 2-(trifluoromethyl)-phenothiazine) as nanomolar, cell-active, and specific Nox1 inhibitors that potently block Nox1-dependent ROS generation, with only marginal activity on other cellular ROS-producing enzymes and receptors including the other Nox isoforms. ML171 also blocks the ROS-dependent formation of ECM-degrading invadopodia in colon cancer cells. Such effects can be reversed by overexpression of Nox1 protein, which is suggestive of a selective mechanism of inhibition of Nox1 by this compound. These results elucidate the relevance of Nox1-dependent ROS generation in mechanisms of cancer invasion and define ML171 as a useful Nox1 chemical probe and potential therapeutic agent for inhibition of cancer cell invasion.
    ACS Chemical Biology 10/2010; 5(10):981-93. · 5.44 Impact Factor
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    ABSTRACT: The NADPH oxidase family, consisting of Nox1-5 and Duox1-2, catalyzes the regulated formation of reactive oxygen species (ROS). Highly expressed in the colon, Nox1 needs the organizer subunit NoxO1 and the activator subunit NoxA1 for its activity. The tyrosine kinase c-Src is necessary for the formation of invadopodia, phosphotyrosine-rich structures which degrade the extracellular matrix (ECM). Many Src substrates are invadopodia components, including the novel Nox1 organizer Tks4 and Tks5 proteins. Nox1-dependent ROS generation is necessary for the maintenance of functional invadopodia in human colon cancer cells. However, the signals and the molecular machinery involved in the redox-dependent regulation of invadopodia formation remain unclear. Here, we show that the interaction of NoxA1 and Tks proteins is dependent on Src activity. Interestingly, the abolishment of Src-mediated phosphorylation of Tyr110 on NoxA1 and of Tyr508 on Tks4 blocks their binding and decreases Nox1-dependent ROS generation. The contemporary presence of Tks4 and NoxA1 unphosphorylable mutants blocks SrcYF-induced invadopodia formation and ECM degradation, while the overexpression of Tks4 and NoxA1 phosphomimetic mutants rescues this phenotype. Taken together, these results elucidate the role of c-Src activity on the formation of invadopodia and may provide insight into the mechanisms of tumor formation in colon cancers.
    Molecular biology of the cell 10/2010; 21(23):4287-98. · 5.98 Impact Factor
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    ABSTRACT: Cofilin-actin bundles (rods), which form in axons and den-drites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-de-polymerizing factor (ADF)/cofilin fluorescent proteins by dif-ferential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca 2؉ , and ATP. Cofilin-GFP-containing rods are stable in 500 mM NaCl, whereas rods formed from endogenous proteins are sig-nificantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immuno-fluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physio-logical concentration shows that they are the only proteins nec-essary for rod formation. Cytoplasmic rod formation is inhib-ited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultra-structural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin. Microischemia (mimicked by transient ATP depletion), oxi-dative stress (mimicked by peroxide or NO), excessive gluta-mate or AMPA 4 stimulation, and small soluble forms of amy-loid ␤ peptide (A␤-(1– 42)) cause within neurites of cultured hippocampal or cortical neurons the formation of rodlike inclu-sions (rods) composed of actin and the actin assembly-regula-tory proteins, actin-depolymerizing factor (ADF) and/or cofilin (1–3). These neuronal treatments induce the dephosphoryla-tion (activation) of ADF/cofilin in neurons in which rods form. Neuronal rods failed to stain with fluorescent derivatives of phalloidin, a mushroom toxin widely used to identify filamen-tous actin (F-actin) structures (4). However, ADF/cofilins bind to a slightly twisted form of F-actin in which the phalloidin binding site is eliminated (5, 6), suggesting that rods might be composed of ADF/cofilin-saturated F-actin. ADF/cofilin-containing rods and aggregates were also observed in hippocampal neurons treated with Pak (p21-activated kinase) inhibitor, PAK18 (7). Down-regulation of Pak activity occurs in human Alzheimer disease (AD) brain and correlates with cogni-tive deficits in AD mice (7). Rod-shaped cofilin-immunostained structures were found in human AD brains but not in human con-trol brain (1) and can be induced in organotypic hippocampal cul-tures (8). Significantly, brains from perfusion-fixed transgenic mice expressing human amyloid precursor protein with familial AD mutations also contain rods (2, 7), demonstrating that rods are not post-mortem artifacts. Mature rods can completely occlude the neurite, blocking transport and causing distal atrophy and syn-aptic dysfunction (1, 2, 9). Actin-containing rodlike structures are not restricted to neu-rons. They were first identified in the nucleus of cultured Dic-tyostelium and HeLa cells treated with high concentrations of DMSO (10); DMSO-induced rods were later shown to contain cofilin (11). Actin-containing rods also have been identified in the nucleus of muscle cells of patients with nemaline myopathy (12). Expression in cultured cells of human skeletal muscle actin containing a nemaline myopathy mutation results in for-mation of nuclear rods, only some of which stain for cofilin (13). Therefore, there are multiple ways to form rod-shaped actin inclu-sions with different compositions. Thus, knowing the components of rods is an important step in understanding the mechanism of their formation and in finding ways to inhibit or reverse their for-mation in neurons before permanent damage ensues.
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    [Show abstract] [Hide abstract]
    ABSTRACT: Cofilin-actin bundles (rods), which form in axons and den-drites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-de-polymerizing factor (ADF)/cofilin fluorescent proteins by dif-ferential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca 2؉ , and ATP. Cofilin-GFP-containing rods are stable in 500 mM NaCl, whereas rods formed from endogenous proteins are sig-nificantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immuno-fluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physio-logical concentration shows that they are the only proteins nec-essary for rod formation. Cytoplasmic rod formation is inhib-ited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultra-structural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin. Microischemia (mimicked by transient ATP depletion), oxi-dative stress (mimicked by peroxide or NO), excessive gluta-mate or AMPA 4 stimulation, and small soluble forms of amy-loid ␤ peptide (A␤-(1– 42)) cause within neurites of cultured hippocampal or cortical neurons the formation of rodlike inclu-sions (rods) composed of actin and the actin assembly-regula-tory proteins, actin-depolymerizing factor (ADF) and/or cofilin (1–3). These neuronal treatments induce the dephosphoryla-tion (activation) of ADF/cofilin in neurons in which rods form. Neuronal rods failed to stain with fluorescent derivatives of phalloidin, a mushroom toxin widely used to identify filamen-tous actin (F-actin) structures (4). However, ADF/cofilins bind to a slightly twisted form of F-actin in which the phalloidin binding site is eliminated (5, 6), suggesting that rods might be composed of ADF/cofilin-saturated F-actin. ADF/cofilin-containing rods and aggregates were also observed in hippocampal neurons treated with Pak (p21-activated kinase) inhibitor, PAK18 (7). Down-regulation of Pak activity occurs in human Alzheimer disease (AD) brain and correlates with cogni-tive deficits in AD mice (7). Rod-shaped cofilin-immunostained structures were found in human AD brains but not in human con-trol brain (1) and can be induced in organotypic hippocampal cul-tures (8). Significantly, brains from perfusion-fixed transgenic mice expressing human amyloid precursor protein with familial AD mutations also contain rods (2, 7), demonstrating that rods are not post-mortem artifacts. Mature rods can completely occlude the neurite, blocking transport and causing distal atrophy and syn-aptic dysfunction (1, 2, 9). Actin-containing rodlike structures are not restricted to neu-rons. They were first identified in the nucleus of cultured Dic-tyostelium and HeLa cells treated with high concentrations of DMSO (10); DMSO-induced rods were later shown to contain cofilin (11). Actin-containing rods also have been identified in the nucleus of muscle cells of patients with nemaline myopathy (12). Expression in cultured cells of human skeletal muscle actin containing a nemaline myopathy mutation results in for-mation of nuclear rods, only some of which stain for cofilin (13). Therefore, there are multiple ways to form rod-shaped actin inclu-sions with different compositions. Thus, knowing the components of rods is an important step in understanding the mechanism of their formation and in finding ways to inhibit or reverse their for-mation in neurons before permanent damage ensues.
    Journal of Biological Chemistry 01/2010; · 4.60 Impact Factor
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    ABSTRACT: NADPH oxidase 1 (NOX1) is highly expressed in colon epithelial cells, where it generates reactive oxygen species (ROS) to interact with normal and pathogenic bacteria. Excessive reactive ROS production is associated with damage to the intestinal mucosa, particularly in mucosal lesions of inflammatory bowel disease (IBD). Studies have shown that NOX1 levels are increased in human prostate cancer, and might also play a role in angiogenesis, cell growth, and tumor pathogenesis. The identification of potent, selective inhibitors of NOX1 may lead to potential therapeutic candidates for excess cell proliferation, cancer, and IBD. This project demonstrated that the molecular probe ML090 (CID-616479) is neither a hydrogen peroxide scavenger, nor a general cell toxin on the time scale of cellular NOX inhibition assays. The specificity of the probe for NOX1 over NOX2, 3 and 4 in a 293 assay system suggests that a target specific to the NOX1 system is the molecular target. ML090 should serve as a useful probe for cellular systems where inhibition of NOX1, and not other members of the NOX family, is desired. This compound provides a significant improvement over the previously existing non-selective NOX inhibitor, diphenylene iodium.
    Probe Reports from the NIH Molecular Libraries Program, 01/2010; National Center for Biotechnology Information (US).
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    ABSTRACT: Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca(2+), and ATP. Cofilin-GFP-containing rods are stable in 500 mM NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.
    Journal of Biological Chemistry 12/2009; 285(8):5450-60. · 4.60 Impact Factor
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    ABSTRACT: The mechanisms that determine localized formation of reactive oxygen species (ROS) through NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (Nox) family members in nonphagocytic cells are unknown. We show that the c-Src substrate proteins Tks4 (tyrosine kinase substrate with four SH3 domains) and Tks5 are functional members of a p47(phox)-related organizer superfamily. Tks proteins selectively support Nox1 and Nox3 (and not Nox2 and Nox4) activity in reconstituted cellular systems and interact with the NoxA1 activator protein through an Src homology 3 domain-mediated interaction. Endogenous Tks4 is required for Rac guanosine triphosphatase- and Nox1-dependent ROS production by DLD1 colon cancer cells. Our results are consistent with the Tks-mediated recruitment of Nox1 to invadopodia that form in DLD1 cells in a Tks- and Nox-dependent fashion. We propose that Tks organizers represent previously unrecognized members of an organizer superfamily that link Nox to localized ROS formation.
    Science Signaling 09/2009; 2(88):ra54. · 7.65 Impact Factor
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    ABSTRACT: Chronic myeloid leukemia (CML) is a lethal hematological disorder caused by the p210(Bcr-Abl) oncogene. Previous studies have suggested that p210(Bcr-Abl) transformation contributes to homing and retention defects, typical of immature myeloid cells in CML, by attenuating chemotactic response to stromal-derived factor-1alpha (SDF-1alpha). As Rho family GTPases are key regulators of the cytoskeleton and have been previously found to interact with p210(Bcr-Abl), this study aimed to determine whether p210(Bcr-Abl) signaling affects SDF-1alpha chemotaxis through Rho GTPase signaling. We found that SDF-1alpha stimulated Cdc42 GTPase activation in myeloid progenitor 32D, but not in p210(Bcr-Abl)-transformed (32Dp210) cells. In fact, the basal level of active Cdc42 was elevated in 32Dp210 cells and mononuclear cells isolated from bone marrow of CML patients. Inhibition of p210(Bcr-Abl) kinase activity decreased basal Cdc42 activity and restored SDF-1alpha-induced Cdc42 and migration responses. Transduction of active Tat-Cdc42V12 abolished this reconstituted chemotactic response. As Cdc42 is particularly important in cytoskeletal remodeling and directional sensing, these results suggest that sustained activation of Cdc42 GTPase through p210(Bcr-Abl) tyrosine kinase signaling in CML cells contributes to defects in SDF-1alpha-chemotactic response due to desensitization of the actin polarization signal required for directional migration.
    Oncogene 09/2009; 28(46):4105-15. · 8.56 Impact Factor
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    ABSTRACT: Rac1 and Rac2, members of the small Rho GTPase family, play essential roles in coordinating directional migration and superoxide production during neutrophil responses to chemoattractants. Although earlier studies in Rac1 and Rac2 knockout mice have demonstrated unique roles for each Rac isoform in chemotaxis and NADPH oxidase activation, it is still unclear how human neutrophils use Rac1 and Rac2 to achieve their immunological responses to foreign agent stimulation. In the current study, we used TAT dominant-negative Rac1-T17N and Rac2-T17N fusion proteins to acutely alter the activity of Rac1 and Rac2 individually in human neutrophils. We demonstrate distinct activation kinetics and different roles for Rac1 and Rac2 in response to low vs high concentrations of fMLP. These observations were verified using neutrophils from mice in which Rac1 or Rac2 was genetically absent. Based on these results, we propose a model to explain how human neutrophils kill invading microbes while limiting oxidative damage to the adjacent surrounding healthy tissue through the differential activation of Rac1 and Rac2 in response to different concentrations of chemoattractant.
    The Journal of Immunology 09/2009; 183(4):2718-28. · 5.36 Impact Factor
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    ABSTRACT: Cell migration involves the cooperative reorganization of the actin and microtubule cytoskeletons, as well as the turnover of cell-substrate adhesions, under the control of Rho family GTPases. RhoA is activated at the leading edge of motile cells by unknown mechanisms to control actin stress fiber assembly, contractility, and focal adhesion dynamics. The microtubule-associated guanine nucleotide exchange factor (GEF)-H1 activates RhoA when released from microtubules to initiate a RhoA/Rho kinase/myosin light chain signaling pathway that regulates cellular contractility. However, the contributions of activated GEF-H1 to coordination of cytoskeletal dynamics during cell migration are unknown. We show that small interfering RNA-induced GEF-H1 depletion leads to decreased HeLa cell directional migration due to the loss of the Rho exchange activity of GEF-H1. Analysis of RhoA activity by using a live cell biosensor revealed that GEF-H1 controls localized activation of RhoA at the leading edge. The loss of GEF-H1 is associated with altered leading edge actin dynamics, as well as increased focal adhesion lifetimes. Tyrosine phosphorylation of focal adhesion kinase and paxillin at residues critical for the regulation of focal adhesion dynamics was diminished in the absence of GEF-H1/RhoA signaling. This study establishes GEF-H1 as a critical organizer of key structural and signaling components of cell migration through the localized regulation of RhoA activity at the cell leading edge.
    Molecular biology of the cell 08/2009; 20(18):4070-82. · 5.98 Impact Factor
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    Jun-Sub Kim, Timothy Y Huang, Gary M Bokoch
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    ABSTRACT: Cellular stimuli generate reactive oxygen species (ROS) via the local action of NADPH oxidases (Nox) to modulate cytoskeletal organization and cell migration through unknown mechanisms. Cofilin is a major regulator of cellular actin dynamics whose activity is controlled by phosphorylation/dephosphorylation at Ser3. Here we show that Slingshot-1L (SSH-1L), a selective cofilin regulatory phosphatase, is involved in H(2)O(2)-induced cofilin dephosphorylation and activation. SSH-1L is activated by its release from a regulatory complex with 14-3-3zeta protein through the redox-mediated oxidation of 14-3-3zeta by H(2)O(2). The ROS-dependent activation of the SSH-1L-cofilin pathway stimulates the SSH-1L-dependent formation of cofilin-actin rods in cofilin-GFP-expressing HeLa cells. Similarly, the formation of endogenous ROS stimulated by angiotensin II (AngII) also activates the SSH-1L-cofilin pathway via oxidation of 14-3-3zeta to increase AngII-induced membrane ruffling and cell motility. These results suggest that the formation of ROS by NADPH oxidases engages a SSH-1L-cofilin pathway to regulate cytoskeletal organization and cell migration.
    Molecular biology of the cell 05/2009; 20(11):2650-60. · 5.98 Impact Factor
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    ABSTRACT: The NADPH oxidase (Nox) enzyme family generates reactive oxygen species (ROS) that contribute to cell signaling, innate immune responses, proliferation, and transcription. The signaling mechanisms that regulate this important enzyme family are only beginning to be understood. Evidence is emerging which suggests that phosphorylation of Nox and/or their regulatory components may be important means of modulating their activity. We describe here the evidence for Nox regulation through the action of kinases, and speculate on how such regulatory mechanisms might contribute to the development of pathological disease states.
    Antioxidants & Redox Signaling 05/2009; 11(10):2429-41. · 8.20 Impact Factor
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    Jun-Sub Kim, Timothy Y Huang, Gary M Bokoch
    [Show abstract] [Hide abstract]
    ABSTRACT: Cellular stimuli generate reactive oxygen species (ROS) via the local action of NADPH oxidases (Nox) to modulate cytoskeletal organization and cell migration through unknown mechanisms. Cofilin is a major regulator of cellular actin dynamics whose activity is controlled by phosphorylation/dephosphorylation at Ser3. Here we show that Slingshot-1L (SSH-1L), a selective cofilin regulatory phosphatase, is involved in H 2 O 2 -induced cofilin dephosphorylation and activa-tion. SSH-1L is activated by its release from a regulatory complex with 14-3-3␨ protein through the redox-mediated oxidation of 14-3-3␨ by H 2 O 2 . The ROS-dependent activation of the SSH-1L-cofilin pathway stimulates the SSH-1L– dependent formation of cofilin-actin rods in cofilin-GFP– expressing HeLa cells. Similarly, the formation of endogenous ROS stimulated by angiotensin II (AngII) also activates the SSH-1L-cofilin pathway via oxidation of 14-3-3␨ to increase AngII-induced membrane ruffling and cell motility. These results suggest that the formation of ROS by NADPH oxidases engages a SSH-1L-cofilin pathway to regulate cytoskeletal organization and cell migration.
  • Source
    Jun-Sub Kim, Timothy Y Huang, Gary M Bokoch
    [Show abstract] [Hide abstract]
    ABSTRACT: Cellular stimuli generate reactive oxygen species (ROS) via the local action of NADPH oxidases (Nox) to modulate cytoskeletal organization and cell migration through unknown mechanisms. Cofilin is a major regulator of cellular actin dynamics whose activity is controlled by phosphorylation/dephosphorylation at Ser3. Here we show that Slingshot-1L (SSH-1L), a selective cofilin regulatory phosphatase, is involved in H 2 O 2 -induced cofilin dephosphorylation and activa-tion. SSH-1L is activated by its release from a regulatory complex with 14-3-3␨ protein through the redox-mediated oxidation of 14-3-3␨ by H 2 O 2 . The ROS-dependent activation of the SSH-1L-cofilin pathway stimulates the SSH-1L– dependent formation of cofilin-actin rods in cofilin-GFP– expressing HeLa cells. Similarly, the formation of endogenous ROS stimulated by angiotensin II (AngII) also activates the SSH-1L-cofilin pathway via oxidation of 14-3-3␨ to increase AngII-induced membrane ruffling and cell motility. These results suggest that the formation of ROS by NADPH oxidases engages a SSH-1L-cofilin pathway to regulate cytoskeletal organization and cell migration.
    Molecular Biology of the Cell. 01/2009; 20:2650-2660.

Publication Stats

19k Citations
1,626.15 Total Impact Points

Institutions

  • 1988–2012
    • The Scripps Research Institute
      • • Department of Immunology and Microbial Science
      • • Department of Cell and Molecular Biology
      • • Department of Molecular and Experimental Medicine
      La Jolla, CA, United States
  • 2009
    • University of Duisburg-Essen
      • Molecular Cell Biology
      Essen, North Rhine-Westphalia, Germany
    • Colorado State University
      • Biochemistry and Molecular Biology
      Fort Collins, CO, United States
  • 1990–2007
    • Montana State University
      • • Department of Immunology and Infectious Diseases
      • • Department of Microbiology
      • • Department of Chemistry & Biochemistry
      Bozeman, MT, United States
  • 2000
    • RWTH Aachen University
      • Institut für Pharmakologie und Toxikologie
      Aachen, North Rhine-Westphalia, Germany
    • Fox Chase Cancer Center
      Philadelphia, Pennsylvania, United States
  • 1999
    • University of Alberta
      • Department of Medicine
      Edmonton, Alberta, Canada
  • 1991–1996
    • Linköping University
      • Faculty of Health Sciences
      Linköping, Östergötland, Sweden
    • Baylor College of Medicine
      • Department of Molecular Physiology & Biophysics
      Houston, TX, United States
  • 1995
    • La Jolla Institute for Allergy & Immunology
      La Jolla, California, United States
  • 1994
    • University of Zurich
      • Physiologisches Institut
      Zürich, ZH, Switzerland