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SH2 Domain (sh2 + domain)
Selected AbstractsSecondary structure assignment of mouse SOCS3 by NMR defines the domain boundaries and identifies an unstructured insertion in the SH2 domainFEBS JOURNAL, Issue 23 2005Jeffrey J. Babon SOCS3 is a negative regulator of cytokine signalling that inhibits Janus kinase-signal transduction and activator of transcription (JAK-STAT) mediated signal tranduction by binding to phosphorylated tyrosine residues on intracellular subunits of various cytokine receptors, as well as possibly the JAK proteins. SOCS3 consists of a short N-terminal sequence followed by a kinase inhibitory region, an extended SH2 domain and a C-terminal suppressor of cytokine signalling (SOCS) box. SOCS3 and the related protein, cytokine-inducible SH2-containing protein, are unique among the SOCS family of proteins in containing a region of mostly low complexity sequence, between the SH2 domain and the C-terminal SOCS box. Using NMR, we assigned and determined the secondary structure of a murine SOCS3 construct. The SH2 domain, unusually, consists of 140 residues, including an unstructured insertion of 35 residues. This insertion fits the criteria for a PEST sequence and is not required for phosphotyrosine binding, as shown by isothermal titration calorimetry. Instead, we propose that the PEST sequence has a functional role unrelated to phosphotyrosine binding, possibly mediating efficient proteolytic degradation of the protein. The latter half of the kinase inhibitory region and the entire extended SH2 subdomain form a single ,-helix. The mapping of the true SH2 domain, and the location of its C terminus more than 50 residues further downstream than predicted by sequence homology, explains a number of previously unexpected results that have shown the importance of residues close to the SOCS box for phosphotyrosine binding. [source] CD247 can bind SHC1, no matter if CD247 is phosphorylatedJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2009Tao Liu Abstract On T cell receptor (TCR) stimulation, src homology 2 domain-containing transforming protein C1 (SHC1) had been found to bind the tyrosine-phosphorylated CD247 chain of the receptor via its src homology 2 (SH2) domain, delivering signals that control T cell development and activation. However, how the phosphorylation of CD247 led to the instant binding has not been characterized clearly. To study the binding process in detail, we simulated and compared the interaction processes of the SH2 domain with CD247 and phosphorylated CD247, respectively. Unexpectedly, the simulation revealed that SHC1 can also bind the nonphosphorylated CD247 peptide, which was further validated to be a weak binding by affinity pull-down experiment. The molecular dynamics (MD) simulation also revealed that the CD247 peptide formed a folding conformation with its Leu209 inserted into the hydrophobic binding pocket in SHC1. And on phosphorylation, it was the electrostatic attraction between the CD247 Tyr(P)206 and the SHC1 Tyr(P)-binding pocket that destroyed the folding conformation of the nonphosphorylated CD247 and, aided by the electrostatic attraction between SHC1 and the Asp203 of CD247, led to the extended conformation of the phosphorylated CD247 binding to SHC1 strongly. The results suggest that nonphosphorylated CD247 can recruit SHC1 in advance to prepare for the instant needs for SHC1 on TCR stimulation. In view of the ubiquity of phosphorylation in protein interaction regulation, we think this study also exemplified the usefulness of MD in more interactome research involving phosphorylation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Csk-homologous kinase interacts with SHPS-1 and enhances neurite outgrowth of PC12 cellsJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Hiroaki Mitsuhashi Abstract SHPS-1 is an immunoglobulin superfamily protein with four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic region. Various neurotrophic factors induce the tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with the protein tyrosine phosphatase SHP-2. Using a yeast two-hybrid screen, we identified a protein tyrosine kinase, Csk-homologous kinase (CHK), as an SHPS-1-interacting protein. Immunoprecipitation and pull-down assays using glutathione S -transferase (GST) fusion proteins containing the Src homology 2 (SH2) domain of CHK revealed that CHK associates with tyrosine-phosphorylated SHPS-1 via its SH2 domain. HIS3 assay in a yeast two-hybrid system using the tyrosine-to-phenylalanine mutants of SHPS-1 indicated that the first and second ITIMs of SHPS-1 are required to bind CHK. Over-expression of wild-type CHK, but not a kinase-inactive CHK mutant, enhanced the phosphorylation of SHPS-1 and its subsequent association with SHP-2. CHK phosphorylated each of four tyrosines in the cytoplasmic region of SHPS-1 in vitro. Co-expression of SHPS-1 and CHK enhanced neurite outgrowth in PC12 cells. Thus, CHK phosphorylates and associates with SHPS-1 and is involved in neural differentiation via SHP-2 activation. [source] Fyn is a novel target of (,)-epigallocatechin gallate in the inhibition of JB6 Cl41 cell transformation,MOLECULAR CARCINOGENESIS, Issue 3 2008Zhiwei He Abstract The cancer preventive action of (,)-epigallocatechin gallate (EGCG), found in green tea, is strongly supported by epidemiology and laboratory research data. However, the mechanism by which EGCG inhibits carcinogenesis and cell transformation is not clear. In this study, we report that EGCG suppressed epidermal growth factor (EGF)-induced cell transformation in JB6 cells. We also found that EGCG inhibited EGF-induced Fyn kinase activity and phosphorylation in vitro and in vivo. Fyn was implicated in the process because EGF-induced JB6 cell transformation was inhibited by small interfering RNA (siRNA)-Fyn-JB6 cells. With an in vitro protein-binding assay, we found that EGCG directly bound with the GST-Fyn-SH2 domain but not the GST-Fyn-SH3 domain. The Kd value for EGCG binding to the Fyn SH2 domain was 0.367,±,0.122 µM and Bmax was 1.35,±,0.128 nmol/mg. Compared with control JB6 Cl41 cells, EGF-induced phosphorylation of p38 MAP kinase (p38 MAPK) (Thr180/Tyr182), ATF-2 (Thr71) and signal transducer and activator of transcription 1 (STAT1) (Thr727) was decreased in siRNA-Fyn-JB6 cells. EGCG could inhibit the phosphorylation of p38 MAPK, ATF-2, and STAT1. The DNA binding ability of AP-1, STAT1, and ATF-2 was also decreased in siRNA-Fyn-JB6 cells. Overall, these results demonstrated that EGCG interacted with Fyn and inhibited Fyn kinase activity and thereby regulated EGF-induced cell transformation. Inhibition of Fyn kinase activity is a novel and important mechanism that may be involved in EGCG-induced inhibition of cell transformation. © 2007 Wiley-Liss, Inc. [source] The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain bindingPROTEIN SCIENCE, Issue 4 2007Shugui Chen Abstract The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis -interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain. [source] Low free energy cost of very long loop insertions in proteinsPROTEIN SCIENCE, Issue 2 2003Michelle Scalley-Kim Abstract Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 × 108 sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60,80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures. [source] Regulation of the epididymal receptor tyrosine kinase ros by the protein tyrosine phosphatase SHP-1AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2002Rico Pusch The SH2 domain PTP SHP-1 was recently identified as a potent negative regulator of the orphan receptor tyrosine kinase Ros, an important regulator of epidimys differentiation (Keilhack et al. J Cell Biol 2001; 152:325,334). Phosphorylated Ros strongly and directly associates with SHP-1 in yeast-two-hybrid, GST pull-down, and coimmunoprecipitation experiments. Catalytically inactive SHP-1C455S exhibits greatly elevated binding to phosphorylated Ros. Direct Ros,SHP-1 interaction is mediated by the SHP-1 N-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively down-regulates Ros-dependent proliferation and transformation. Elevated phosphorylation of Ros in ,viable motheaten (me-v)' mice which, have strongly reduced SHP-1 activity, suggests that Ros signaling is under control of SHP-1 in vivo. Thus sterility of male me-v mice seems to be related to dysregulation of Ros. A synthetic phosphopeptide derived from the Ros sequence around Y2267 potently activates recombinant SHP-1 in vitro but is not a good substrate for SHP-1. In contrast, phosphorylation sites in the activation loop of Ros are effectively dephosphorylated. Based on these observations we propose a mechanistic model of Ros,SHP-1 interaction. Using fusion proteins of SHP-1 variants and of Ros with GFP-proteins of different spectral characteristics the interaction of Ros and SHP-1 can be visualized in intact cells by different microscopic techniques. [source] Preliminary crystallographic characterization of the Grb2 SH2 domain in complex with a FAK-derived phosphotyrosyl peptideACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010Hsiao-Hsin Chen Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein with a single SH2 domain that specifically binds to focal adhesion kinase (FAK) when residue Tyr925 of FAK is phosphorylated. The Grb2,FAK interaction is associated with cellular integrin-activated signal transduction events leading to the activation of the Ras-MAPK pathway. Crystals of the Grb2 SH2 domain in complex with a phosphopeptide corresponding to residues 921,930 of FAK have been obtained using the sitting-drop vapour-diffusion technique. The crystals belonged to space group P3121, with unit-cell parameters a = b = 102.7, c = 127.6,Å, , = , = 90.0, , = 120.0°. A diffraction data set was collected from a flash-cooled crystal at 100,K to 2.49,Å resolution using synchrotron radiation. Structure determination by molecular replacement and analysis of the detailed structure of the complex are currently in progress. [source] Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responsesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2008Manchao Zhang Abstract The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin. J. Cell. Biochem. 103: 162,181, 2008. © 2007 Wiley-Liss, Inc. [source] An electrostatic network and long-range regulation of Src kinasesPROTEIN SCIENCE, Issue 11 2008Elif Ozkirimli Abstract The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain,domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains. [source] The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain bindingPROTEIN SCIENCE, Issue 4 2007Shugui Chen Abstract The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis -interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain. [source] Anaplasma phagocytophilum AnkA is tyrosine-phosphorylated at EPIYA motifs and recruits SHP-1 during early infectionCELLULAR MICROBIOLOGY, Issue 5 2007Jacob W. IJdo Summary Anaplasma phagocytophilum is an intracellular pathogen that infects and survives in neutrophilic granulocytes. The A. phagocytophilum genome encodes a type four secretion system (T4SS) that may facilitate intracellular survival by translocation of virulence factors, but to date, no such factors have been identified. Because T4SS-translocated proteins of several intracellular organisms undergo tyrosine phosphorylation by host cell kinases, we investigated tyrosine phosphorylation of A. phagocytophilum proteins during infection. Within minutes after incubation of A. phagocytophilum with HL-60 cells or PMN, a 190 kDa bacterial protein, AnkA, was increasingly tyrosine-phosphorylated. A. phagocytophilum binding to host cells without entry was sufficient for AnkA tyrosine phosphorylation. An in vitro Src kinase assay demonstrated that purified AnkA expressed in Escherichia coli was phosphorylated at tyrosines located at the C-terminal portion of AnkA. Similarly, AnkA expressed in COS-7 cells underwent tyrosine phosphorylation by Src at the C-terminus. The phosphorylated tyrosines were located in EPIYA motifs that display the consensus sequence for binding to SH2 domains. Immunoprecipitation studies demonstrated AnkA binding to the host cell phosphatase SHP-1 during early infection. Phosphorylation of the EPIYA motifs and the presence of the SH2 domains were necessary for AnkA,SHP-1 interaction. We conclude that AnkA is a translocated virulence factor that is tyrosine-phosphorylated by host cell kinases upon translocation into the host cell early during infection. A. phagocytophilum may manipulate the host cell through SHP-1 recruitment. [source] Utilization of a Common Pathway for the Synthesis of High Affinity Macrocyclic Grb2 SH2 Domain-Binding Peptide Mimetics That Differ in the Configuration at One Ring JunctionCHEMISTRY & BIODIVERSITY, Issue 4 2005Zhen-Dan Shi As typified by 2-{(9S,10S,14R,18S)-18-(2-amino-2-oxoethyl)-14-[(5-methyl-1H -indol-1-yl)methyl]-8,17,20-trioxo-10-[4-(phosphonomethyl)phenyl]-7,16,19-triazaspiro[5.14]icos-11-en-9-yl}acetic acid ((14R)- 1b), ring-closing methathesis-derived macrocyclic tetrapeptide mimetics have recently been reported that bind with high affinity to Grb2 SH2 domains in both extracellular and whole-cell assays. The synthetic complexity of this class of agents limits further therapeutic development. Although a significant component of this synthetic complexity arises from the presence of three stereogenic centers, C(9) (S), C(10) (S), and C(14) (R), it is unclear whether stereoselective introduction of defined configuration at C(14) is required for high-affinity binding. Reported herein is a synthetic route to these macrocycles lacking stereoselectivity in the formation of the C(14) ring junction, which is four synthetic steps shorter than the original stereoselective synthesis. Separation of C(14)-epimers obtained by this approach was achieved by preparative HPLC. Molecular-dynamics studies of ligands bound to the Grb2 SH2 domain protein indicated that the (14R)-configuration should display more-favorable interactions with the protein relative to the (14S)-epimer. Indeed, although surface-plasmon-resonance-derived binding constants to Grb2 SH2 domain protein indicated that the affinity of the (14R)-epimer (KD=4.8,nM) is greater than that of the (14S)-epimer (KD=11,nM), it is only marginally so. Therefore, little affinity would be lost through a non-stereoselective synthesis of the C(14)-center. Further studies are in progress to explore reduced structural complexity at the C(14)-center. [source] | ||||||||||||||||