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David B. Sacks - One of the best experts on this subject based on the ideXlab platform.
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IQGAP1 binds AMPK and is required for maximum AMPK activation
The Journal of biological chemistry, 2020Co-Authors: Andrew C Hedman, Chase J Morgan, Laetitia Gorisse, Swetha Parvathaneni, David B. SacksAbstract:AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.
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Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function.
The Journal of biological chemistry, 2020Co-Authors: Andrew C Hedman, Laetitia Gorisse, Roland S Annan, Dean E. Mcnulty, David B. SacksAbstract:IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.
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ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the rho gtpase cdc42
Journal of Biological Chemistry, 2020Co-Authors: Laetitia Gorisse, Andrew C Hedman, Zhigang Li, Craig D Wagner, David K Worthylake, Francesca Zappacosta, Roland S Annan, David B. SacksAbstract:: IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475 and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did wild-type IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than wild-type IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
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ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the rho gtpase cdc42
Journal of Biological Chemistry, 2020Co-Authors: Laetitia Gorisse, Andrew C Hedman, Craig D Wagner, David K Worthylake, Francesca Zappacosta, Roland S Annan, David B. SacksAbstract:IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
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Endothelial IQGAP1 regulates leukocyte transmigration by directing the LBRC to the site of diapedesis.
The Journal of experimental medicine, 2019Co-Authors: David P. Sullivan, Prarthana Dalal, Fanny Jaulin, David B. Sacks, Geri Kreitzer, William A. MullerAbstract:Transendothelial migration (TEM) of leukocytes across the endothelium is critical for inflammation. In the endothelium, TEM requires the coordination of membrane movements and cytoskeletal interactions, including, prominently, recruitment of the lateral border recycling compartment (LBRC). The scaffold protein IQGAP1 was recently identified in a screen for LBRC-interacting proteins. Knockdown of endothelial IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. Expression of truncated IQGAP1 constructs demonstrated that the calponin homology domain is required for IQGAP1 localization to endothelial borders and that the IQ domain, on the same IQGAP1 polypeptide, is required for its function in TEM. This is the first reported function of IQGAP1 requiring two domains to be present on the same polypeptide. Additionally, we show for the first time that IQGAP1 in the endothelium is required for efficient TEM in vivo. These findings reveal a novel function for IQGAP1 and demonstrate that IQGAP1 in endothelial cells facilitates TEM by directing the LBRC to the site of TEM.
Andrew C Hedman - One of the best experts on this subject based on the ideXlab platform.
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IQGAP1 binds AMPK and is required for maximum AMPK activation
The Journal of biological chemistry, 2020Co-Authors: Andrew C Hedman, Chase J Morgan, Laetitia Gorisse, Swetha Parvathaneni, David B. SacksAbstract:AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.
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Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function.
The Journal of biological chemistry, 2020Co-Authors: Andrew C Hedman, Laetitia Gorisse, Roland S Annan, Dean E. Mcnulty, David B. SacksAbstract:IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.
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ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the rho gtpase cdc42
Journal of Biological Chemistry, 2020Co-Authors: Laetitia Gorisse, Andrew C Hedman, Craig D Wagner, David K Worthylake, Francesca Zappacosta, Roland S Annan, David B. SacksAbstract:IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
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ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the rho gtpase cdc42
Journal of Biological Chemistry, 2020Co-Authors: Laetitia Gorisse, Andrew C Hedman, Zhigang Li, Craig D Wagner, David K Worthylake, Francesca Zappacosta, Roland S Annan, David B. SacksAbstract:: IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475 and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did wild-type IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than wild-type IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
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endogenous IQGAP1 and iqgap3 do not functionally interact with ras
Scientific Reports, 2019Co-Authors: Chase J Morgan, Andrew C Hedman, David B. SacksAbstract:The Ras family of small GTPases modulates numerous essential processes. Activating Ras mutations result in hyper-activation of selected signaling cascades, which leads to human diseases. The high frequency of Ras mutations in human malignant neoplasms has led to Ras being a desirable chemotherapeutic target. The IQGAP family of scaffold proteins binds to and regulates multiple signaling molecules, including the Rho family GTPases Rac1 and Cdc42. There are conflicting data in the published literature regarding interactions between IQGAP and Ras proteins. Initial reports showed no binding, but subsequent studies claim associations of IQGAP1 and IQGAP3 with K-Ras and H-Ras, respectively. Therefore, we set out to resolve this controversy. Here we demonstrate that neither endogenous IQGAP1 nor endogenous IQGAP3 binds to the major Ras isoforms, namely H-, K-, and N-Ras. Importantly, Ras activation by epidermal growth factor is not altered when IQGAP1 or IQGAP3 proteins are depleted from cells. These data strongly suggest that IQGAP proteins are not functional interactors of H-, K-, or N-Ras and challenge the rationale for targeting the interaction of Ras with IQGAP for the development of therapeutic agents.
George S. Bloom - One of the best experts on this subject based on the ideXlab platform.
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actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain
Cytoskeleton, 2004Co-Authors: Scott C. Mateer, Leah E. Morris, Damond A. Cromer, Lorena B. Benseñor, George S. BloomAbstract:IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, β-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5–50 μM Kd) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP12-210, which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP12-210 was found to be monomeric, to bind F-actin with a Kd of ∼47 μM, to saturate F-actin at a molar ratio of one IQGAP12-210 per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD. Cell Motil. Cytoskeleton 58:231–241, 2004. © 2004 Wiley-Liss, Inc.
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Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain.
Cell motility and the cytoskeleton, 2004Co-Authors: Scott C. Mateer, Leah E. Morris, Damond A. Cromer, Lorena B. Benseñor, George S. BloomAbstract:IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, beta-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5-50 microM K(d)) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP1(2-210), which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP1(2-210) was found to be monomeric, to bind F-actin with a K(d) of approximately 47 microM, to saturate F-actin at a molar ratio of one IQGAP1(2-210) per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD.
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the mechanism for regulation of the f actin binding activity of IQGAP1 by calcium calmodulin
Journal of Biological Chemistry, 2002Co-Authors: Scott C. Mateer, Damond A. Cromer, Amanda E. Mcdaniel, Valérie Nicolas, Geoffrey Habermacher, Mei-jung Sun Lin, Michelle E. King, George S. BloomAbstract:IQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and β-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca2+ and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAP1 much more efficiently in the presence of Ca2+ than EGTA, and all 8 IQ motifs in each IQGAP1 dimer could bind calmodulin simultaneously. In live cells, IQGAP1-YFP localized to the cell cortex, but elevation of intracellular Ca2+ reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in free intracellular Ca2+ promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments.
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The Mechanism for Regulation of the F-actin Binding Activity of IQGAP1 by Calcium/Calmodulin
The Journal of biological chemistry, 2002Co-Authors: Scott C. Mateer, Damond A. Cromer, Amanda E. Mcdaniel, Valérie Nicolas, Geoffrey Habermacher, Mei-jung Sun Lin, Michelle E. King, George S. BloomAbstract:IQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and β-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca2+ and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAP1 much more efficiently in the presence of Ca2+ than EGTA, and all 8 IQ motifs in each IQGAP1 dimer could bind calmodulin simultaneously. In live cells, IQGAP1-YFP localized to the cell cortex, but elevation of intracellular Ca2+ reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in free intracellular Ca2+ promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments.
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IQGAP1 a rac and cdc42 binding protein directly binds and cross links microfilaments
Journal of Cell Biology, 1997Co-Authors: Anne Marie Bashour, Aaron T Fullerton, Matthew J Hart, George S. BloomAbstract:Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two ∼190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D–sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.
Roland S Annan - One of the best experts on this subject based on the ideXlab platform.
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Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function.
The Journal of biological chemistry, 2020Co-Authors: Andrew C Hedman, Laetitia Gorisse, Roland S Annan, Dean E. Mcnulty, David B. SacksAbstract:IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.
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ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the rho gtpase cdc42
Journal of Biological Chemistry, 2020Co-Authors: Laetitia Gorisse, Andrew C Hedman, Craig D Wagner, David K Worthylake, Francesca Zappacosta, Roland S Annan, David B. SacksAbstract:IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
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ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the rho gtpase cdc42
Journal of Biological Chemistry, 2020Co-Authors: Laetitia Gorisse, Andrew C Hedman, Zhigang Li, Craig D Wagner, David K Worthylake, Francesca Zappacosta, Roland S Annan, David B. SacksAbstract:: IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475 and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did wild-type IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than wild-type IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
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MAPK Scaffold IQGAP1 Binds the EGF Receptor and Modulates Its Activation
The Journal of biological chemistry, 2011Co-Authors: Dean E. Mcnulty, David B. Sacks, Colin D. White, Roland S AnnanAbstract:Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca(2+) attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser(1443), a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.
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IQGAP1 promotes neurite outgrowth in a phosphorylation-dependent manner
The Journal of biological chemistry, 2005Co-Authors: Dean E. Mcnulty, Roland S Annan, Katharine J.m. Marler, Louis Lim, Christine Hall, David B. SacksAbstract:Abstract In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, β-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase Cϵ, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.
Laetitia Gorisse - One of the best experts on this subject based on the ideXlab platform.
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IQGAP1 binds AMPK and is required for maximum AMPK activation
The Journal of biological chemistry, 2020Co-Authors: Andrew C Hedman, Chase J Morgan, Laetitia Gorisse, Swetha Parvathaneni, David B. SacksAbstract:AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.
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Tyrosine phosphorylation of the scaffold protein IQGAP1 in the MET pathway alters function.
The Journal of biological chemistry, 2020Co-Authors: Andrew C Hedman, Laetitia Gorisse, Roland S Annan, Dean E. Mcnulty, David B. SacksAbstract:IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.
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ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the rho gtpase cdc42
Journal of Biological Chemistry, 2020Co-Authors: Laetitia Gorisse, Andrew C Hedman, Craig D Wagner, David K Worthylake, Francesca Zappacosta, Roland S Annan, David B. SacksAbstract:IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
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ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the rho gtpase cdc42
Journal of Biological Chemistry, 2020Co-Authors: Laetitia Gorisse, Andrew C Hedman, Zhigang Li, Craig D Wagner, David K Worthylake, Francesca Zappacosta, Roland S Annan, David B. SacksAbstract:: IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475 and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did wild-type IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than wild-type IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.
