The Experts below are selected from a list of 2583 Experts worldwide ranked by ideXlab platform
Thomas E Smithgall - One of the best experts on this subject based on the ideXlab platform.
-
Subtle Dynamic Changes Accompany HCK Activation by HIV-1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor
Biochemistry, 2015Co-Authors: Thomas E. Wales, Thomas E Smithgall, James M. Hochrein, Christopher R. Morgan, Lori A. Emert-sedlak, John R. EngenAbstract:The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase HCK, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent HCK activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated HCK that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by HCK, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the HCK SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on HCK N-lobe dynamics was completely reversed. These results show that constitutive activation of HCK by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells.
-
interaction with the src homology sh3 sh2 region of the src family kinase HCK structures the hiv 1 nef dimer for kinase activation and effector recruitment
Journal of Biological Chemistry, 2014Co-Authors: Sreya Tarafdar, John Jeff Alvarado, Joanne I Yeh, Thomas E SmithgallAbstract:HIV-1 Nef supports high titer viral replication in vivo and is essential for AIDS progression. Nef function depends on interactions with multiple host cell effectors, including HCK and other Src-family kinases. Here we describe the x-ray crystal structure of Nef in complex with the HCK SH3-SH2 regulatory region to a resolution of 1.86 Å. The complex crystallized as a dimer of complexes, with the conserved Nef PXXPXR motif engaging the HCK SH3 domain. A new intercomplex contact was found between SH3 Glu-93, and Nef Arg-105. Mutagenesis of HCK SH3 Glu-93 interfered with Nef·HCK complex formation and kinase activation in cells. The HCK SH2 domains impinge on the N-terminal region of Nef to stabilize a dimer conformation that exposes Asp-123, a residue critical for Nef function. Our results suggest that in addition to serving as a kinase effector for Nef, HCK binding may reorganize the Nef dimer for functional interaction with other signaling partners.
-
An inhibitor-resistant mutant of HCK protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259
Oncogene, 2008Co-Authors: Teodora Pene-dumitrescu, L F Peterson, N J Donato, Thomas E SmithgallAbstract:Chronic myelogenous leukemia (CML) is driven by Bcr-Abl, a constitutively active protein-tyrosine kinase that stimulates proliferation and survival of myeloid progenitors. Global inhibition of myeloid Src family kinase (SFK) activity with the broad-spectrum pyrrolo-pyrimidine inhibitor, A-419259, blocks proliferation and induces apoptosis in CML cell lines, suggesting that transformation by Bcr-Abl requires SFK activity. However, the contribution of HCK and other individual SFKs to Bcr-Abl signaling is less clear. Here, we developed an A-419259-resistant mutant of HCK by replacing the gatekeeper residue (Thr-338; c-Src numbering) in the inhibitor-binding site with a bulkier methionine residue (HCK-T338M). This substitution reduced HCK sensitivity to A-419259 by more than 30-fold without significantly affecting kinase activity in vitro . Expression of HCK-T338M protected K-562 CML cells and Bcr-Abl-transformed TF-1 myeloid cells from the apoptotic and antiproliferative effects of A-419259. These effects correlated with persistence of HCK-T338M kinase activity in the presence of the compound, and were accompanied by sustained Erk and Stat5 activation. In contrast, control cells expressing equivalent levels of wild-type HCK retained sensitivity to the inhibitor. We also show for the first time that A-419259 induces cell-cycle arrest and apoptosis in primary CD34^+ CML cells with equal potency to imatinib. These data suggest that HCK has a nonredundant function as a key downstream signaling partner for Bcr-Abl and may represent a potential drug target in CML.
-
oligomerization is required for hiv 1 nef induced activation of the src family protein tyrosine kinase HCK
Biochemistry, 2004Co-Authors: Hyunjung Choi, Jerrod A Poe, Thomas E SmithgallAbstract:HCK is a member of the Src protein-tyrosine kinase family and is expressed strongly in macrophages, an important HIV target cell. Previous studies have shown that Nef, an HIV-1 accessory protein essential for AIDS progression, binds and activates HCK through its SH3 domain. Structural analysis suggests that Nef forms oligomers in vivo, which may bring multiple HCK molecules into close proximity and promote autophosphorylation. Using bimolecular GFP fluorescence complementation, we show for the first time that Nef oligomerizes in living cells and that the oligomers localize to the plasma membrane. To test the role of Nef oligomerization in HCK activation, we fused Nef to the hormone-binding domain of the estrogen receptor (Nef-ER), allowing us to control its dimerization with 4-hydroxytamoxifen (4-HT). In Rat-2 fibroblasts co-expressing Nef-ER and HCK, 4-HT treatment induced Nef-ER dimer and tetramer formation, leading to HCK kinase activation and cellular transformation. The number of transformed foci observed with Nef-ER plus HCK in the presence of 4-HT was markedly greater than that observed with wild-type Nef plus HCK, suggesting that enforced oligomerization enhances activation of HCK by Nef in vivo. Enhanced transformation correlated with increased HCK/Nef complex formation at the plasma membrane. In complementary experiments, we observed that a Nef mutant defective for HCK SH3 domain binding (Nef-PA) suppressed HCK kinase activation and transformation by the wild-type HCK/Nef complex. This effect correlated with the formation of a ternary complex between wild-type Nef, Nef-PA, and HCK, suggesting that Nef-PA suppresses HCK activation by blocking wild-type Nef oligomerization. Finally, Nef-ER induced HCK activation in a 4-HT-dependent manner in the macrophage precursor cell line TF-1, suggesting that oligomerization is essential for signaling through HCK in a cell background relevant to HIV infection. Together, these data demonstrate that Nef oligomerization is critical to the activation of HCK in vivo, and suggest that inhibitors of oligomerization may suppress Nef signaling through HCK in HIV-infected macrophages, slowing disease progression.
-
Conserved Residues in the HIV-1 Nef Hydrophobic Pocket are Essential for Recruitment and Activation of the HCK Tyrosine Kinase
Journal of molecular biology, 2004Co-Authors: Hyunjung Choi, Thomas E SmithgallAbstract:The Nef protein of the primate lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is essential for high-titer viral replication and acquired immune deficiency syndrome (AIDS) progression. Nef binds to the macrophage-specific Src family member HCK through its SH3 domain, resulting in constitutive kinase activation capable of transforming rodent fibroblasts. Nef–HCK interaction may be essential for M-tropic HIV replication and AIDS pathogenesis, identifying this virus–host protein complex as a rational target for anti-HIV drug discovery. Here, we investigated whether interaction with HCK is a common feature of Nef alleles from different strains of HIV-1. We compared the ability of four different laboratory HIV-1 Nef alleles (SF2, LAI, ELI, and Consensus) to induce HCK activation and transformation in our Rat-2 fibroblast model. While SF2, LAI, and Consensus Nef all bound and activated HCK, ELI Nef failed to bind to the HCK SH3 domain in vitro and did not cooperate with HCK in fibroblast transformation. Molecular modeling identified three residues in the core region of SF2 Nef (Ala83, His116, and Tyr120) which are substituted in ELI with Glu, Asn, and Ile, respectively. Two of these residues (Ala83 and Tyr120) form part of the hydrophobic pocket that contacts Ile 96 in the RT loop of the HCK SH3 domain in the Nef-SH3 crystal structure. Substitution of SF2 Nef Tyr120 with Ile completely abolished HCK recruitment and activation. In a complementary experiment, substitution of ELI Ile120 with Tyr partly restored ELI Nef-induced HCK activation and transformation in Rat-2 cells. HCK activation increased further by substitution of ELI Glu83 with Ala and Asn116 with His, suggestive of a supportive role for these residues in HCK binding. This study provides the first biological evidence that the HIV-1 Nef hydrophobic pocket is critical to HCK recruitment and activation in vivo. Targeting the Nef hydrophobic pocket with a small molecule may be sufficient to disrupt Nef signaling through HCK in HIV-infected macrophages, slowing disease progression.
Y Yoshihara - One of the best experts on this subject based on the ideXlab platform.
-
superficial zone chondrocytes in normal and osteoarthritic human articular cartilages synthesize novel truncated forms of inter alpha trypsin inhibitor heavy chains which are attached to a chondroitin sulfate proteoglycan other than bikunin
Osteoarthritis and Cartilage, 2008Co-Authors: Y Yoshihara, Barbara Osborn, Arkady Margulis, F Nelson, Matthew C Stewart, Caroline M Milner, Marylin S Rugg, Anna Plaas, K NemotoAbstract:OBJECTIVE: We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IalphaI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids. DESIGN/METHODS: Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR. RESULTS: Bikunin.chondroitin sulfate (CS) and IalphaI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin.CS and full-length (approximately 90 kDa) HCs linked to hyaluronan (HA) as HC.HA (SHAP.HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth. CONCLUSION: The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin.CS and IalphaI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.
-
superficial zone chondrocytes in normal and osteoarthritic human articular cartilages synthesize novel truncated forms of inter alpha trypsin inhibitor heavy chains which are attached to a chondroitin sulfate proteoglycan other than bikunin
Osteoarthritis and Cartilage, 2008Co-Authors: Y Yoshihara, Barbara Osborn, Arkady Margulis, F Nelson, Matthew C Stewart, Caroline M Milner, Marylin S Rugg, Anna Plaas, Anthony J DayAbstract:Summary Objective We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IαI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids. Design/methods Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR. Results Bikunin·chondroitin sulfate (CS) and IαI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin·CS and full-length (∼90kDa) HCs linked to hyaluronan (HA) as HC·HA (SHAP·HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth. Conclusion The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin·CS and IαI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.
Anthony J Day - One of the best experts on this subject based on the ideXlab platform.
-
constitutive expression of inter α inhibitor iαi family proteins and tumor necrosis factor stimulated gene 6 tsg 6 by human amniotic membrane epithelial and stromal cells supporting formation of the heavy chain hyaluronan hc ha complex
Journal of Biological Chemistry, 2012Co-Authors: Suzhen Zhang, Anthony J Day, Scheffer C G TsengAbstract:Abstract Recently we reported HC-HA, a covalent complex formed between heavy chains (HCs) of inter-α-inhibitor (IαI) and hyaluronan (HA) by the catalytic action of tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), is responsible for human amniotic membrane (AM)s anti-inflammatory, anti-scarring and anti-angiogenic actions. At the present time, the only well characterized source of IαI is serum being produced by the liver. This study showed that AM epithelial and stromal cells and stromal matrix all stained positively for HA, HC 1, 2 and 3, bikunin, and TSG-6. TSG-6 mRNA and protein were constitutively expressed by cultured AM epithelial and stromal cells without being upregulated by TNF. In serum-free conditions, these cells expressed IαI, leading to the formation of HC-HA complex that contained both HC1 and HC2. In contrast, only HC1 was found in the HC-HA complex purified from AM. Local production of IαI, the HC-TSG-6 intermediate complex, and HC-HA were abolished when cells were treated with siRNA to HC1, HC2, bikunin (all of which impair the biosynthesis of IαI), or TSG-6 but not to HC3. Collectively, these results indicate that AM is another tissue besides the liver to constitutively produce IαI, and that the HC-HA complex made by this tissue is different from that found at inflammatory sites (e.g. in asthma and arthritis) and in the matrix of the cumulus oocyte complex.
-
superficial zone chondrocytes in normal and osteoarthritic human articular cartilages synthesize novel truncated forms of inter alpha trypsin inhibitor heavy chains which are attached to a chondroitin sulfate proteoglycan other than bikunin
Osteoarthritis and Cartilage, 2008Co-Authors: Y Yoshihara, Barbara Osborn, Arkady Margulis, F Nelson, Matthew C Stewart, Caroline M Milner, Marylin S Rugg, Anna Plaas, Anthony J DayAbstract:Summary Objective We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IαI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids. Design/methods Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR. Results Bikunin·chondroitin sulfate (CS) and IαI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin·CS and full-length (∼90kDa) HCs linked to hyaluronan (HA) as HC·HA (SHAP·HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth. Conclusion The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin·CS and IαI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.
Todd W Miller - One of the best experts on this subject based on the ideXlab platform.
-
biochemical properties of the cdc42 associated tyrosine kinase ack1 substrate specificity autophosphorylation and interaction with HCK
Journal of Biological Chemistry, 2003Co-Authors: Noriko Yokoyama, Todd W MillerAbstract:ACK1 (activated Cdc42-associated kinase 1) is a nonreceptor tyrosine kinase and the only tyrosine kinase known to interact with Cdc42. To characterize the enzymatic properties of ACK, we have expressed and purified active ACK using the baculovirus/Sf9 cell system. This ACK1 construct contains (from N to C terminus) the kinase catalytic domain, SH3 domain, and Cdc42-binding Cdc42/Rac interactive binding (CRIB) domain. We characterized the substrate specificity of ACK1 using synthetic peptides, and we show that the specificity of the ACK1 catalytic domain most closely resembles that of Abl. Purified ACK1 undergoes autophosphorylation, and autophosphorylation enhances kinase activity. We identified Tyr284 in the activation loop of ACK1 as the primary autophosphorylation site using mass spectrometry. When expressed in COS-7 cells, the Y284F mutant ACK1 showed dramatically reduced levels of tyrosine phosphorylation. Although the SH3 and CRIB domains of purified ACK1 are able to bind ligands (a polyproline peptide and Cdc42, respectively), the addition of ligands did not stimulate tyrosine kinase activity. To characterize potential interacting partners for ACK1, we screened several SH2 and SH3 domains for their ability to bind to full-length ACK1 or to the catalytic-SH3-CRIB construct. ACK1 interacts most strongly with the SH3 domains of Src family kinases (Src or HCK) via its C-terminal proline-rich domain. Co-expression of HCK with kinase-inactive ACK1(K158R) in mammalian cells resulted in tyrosine phosphorylation of ACK1, suggesting that ACK1 is a substrate for HCK. Our data suggest that HCK is a novel binding partner for ACK1 that can regulate ACK1 activity by phosphorylation.
-
identification of novel sh3 domain ligands for the src family kinase HCK wiskott aldrich syndrome protein wasp wasp interacting protein wip and elmo1
Journal of Biological Chemistry, 2002Co-Authors: Margaret Porter Scott, Francesca Zappacosta, Eun Young Kim, Roland S Annan, Todd W MillerAbstract:The importance of the SH3 domain of HCK in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of HCK. In this study, we used mass spectrometry to identify approximately 25 potential binding partners for the SH3 domain of HCK from the monocyte cell line U937. Two major interacting proteins were the actin binding proteins Wiskott-Aldrich syndrome protein (WASP) and WASP-interacting protein (WIP). We also focused on a novel interaction between HCK and ELMO1, an 84-kDa protein that was recently identified as the mammalian ortholog of the Caenorhabditis elegans gene, ced-12. In mammalian cells, ELMO1 interacts with Dock180 as a component of the CrkII/Dock180/Rac pathway responsible for phagocytosis and cell migration. Using purified proteins, we confirmed that WASP-interacting protein and ELMO1 interact directly with the SH3 domain of HCK. We also show that HCK and ELMO1 interact in intact cells and that ELMO1 is heavily tyrosine-phosphorylated in cells that co-express HCK, suggesting that it is a substrate of HCK. The binding of ELMO1 to HCK is specifically dependent on the interaction of a polyproline motif with the SH3 domain of HCK. Our results suggest that these proteins may be novel activators/effectors of HCK.
-
reciprocal regulation of HCK activity by phosphorylation of tyr527 and tyr416 effect of introducing a high affinity intramolecular sh2 ligand
Journal of Biological Chemistry, 2000Co-Authors: John Kuriyan, Margaret Porter, Thomas Schindler, Todd W MillerAbstract:The Src family tyrosine kinase HCK possesses two phosphorylation sites, Tyr527 and Tyr416, that affect the catalytic activity in opposite ways. When phosphorylated, Tyr527 and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this sequence does not conform to the sequence of the high affinity SH2 ligand, pYEEI. We mutated this sequence to YEEI and show that this mutant form of HCK cannot be activated by exogenous SH2 ligands. The SH3 domain of HCK is also involved in an inhibitory interaction with the catalytic domain. The SH3 ligand Nef binds to and activates YEEI-HCK mutant in a similar manner to wild-type HCK, indicating that disrupting the SH3 interaction overrides the strengthened SH2 interaction. The other phosphorylation site, Tyr416, is the autophosphorylation site in the activation loop. Phosphorylation of Tyr416 is required for HCK activation. We mutated this residue to alanine and characterized its catalytic activity. The Y416A mutant shows a higherK m value for peptide and a lowerV max than autophosphorylated wild-type HCK. We also present evidence for cross-talk between the activation loop and the intramolecular binding of the SH2 and SH3 domains.
K Nemoto - One of the best experts on this subject based on the ideXlab platform.
-
superficial zone chondrocytes in normal and osteoarthritic human articular cartilages synthesize novel truncated forms of inter alpha trypsin inhibitor heavy chains which are attached to a chondroitin sulfate proteoglycan other than bikunin
Osteoarthritis and Cartilage, 2008Co-Authors: Y Yoshihara, Barbara Osborn, Arkady Margulis, F Nelson, Matthew C Stewart, Caroline M Milner, Marylin S Rugg, Anna Plaas, K NemotoAbstract:OBJECTIVE: We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IalphaI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids. DESIGN/METHODS: Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR. RESULTS: Bikunin.chondroitin sulfate (CS) and IalphaI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin.CS and full-length (approximately 90 kDa) HCs linked to hyaluronan (HA) as HC.HA (SHAP.HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth. CONCLUSION: The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin.CS and IalphaI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.
