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1
Panchamoorthy, G., T. Fukazawa, L. Stolz, G. Payne, K. Reedquist, S. Shoelson, Z. Songyang, L. Cantley, C. Walsh, and H. Band. "Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn." Molecular and Cellular Biology 14, no. 9 (September 1994): 6372–85. http://dx.doi.org/10.1128/mcb.14.9.6372-6385.1994.
Full textAbstract:
The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.
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Panchamoorthy, G., T. Fukazawa, L. Stolz, G. Payne, K. Reedquist, S. Shoelson, Z. Songyang, L. Cantley, C. Walsh, and H. Band. "Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn." Molecular and Cellular Biology 14, no. 9 (September 1994): 6372–85. http://dx.doi.org/10.1128/mcb.14.9.6372.
Full textAbstract:
The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.
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Richard, S., D. Yu, K. J. Blumer, D. Hausladen, M. W. Olszowy, P. A. Connelly, and A. S. Shaw. "Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1." Molecular and Cellular Biology 15, no. 1 (January 1995): 186–97. http://dx.doi.org/10.1128/mcb.15.1.186.
Full textAbstract:
src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.
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Dumas, Caroline, Anna Schmoker, Shannon Bennett, Amara Chittenden, Chelsea Darwin, Helena Gaffney, Hannah Lewis, et al. "Novel Interactors of the SH2 Domain of the Signaling Adaptors CRK and CRKL Identified in Neuro2A Cells." American Journal of Undergraduate Research 19, no. 3 (December 31, 2022): 47–55. http://dx.doi.org/10.33697/ajur.2022.068.
Full textAbstract:
CT10 regulator of kinase (CRK) and CRK-like (CRKL) form a family of signaling adaptor proteins that serve important roles in the regulation of fundamental cellular processes, including cell motility and proliferation, in a variety of cell types. The Src Homology 2 (SH2) domain of CRK and CRKL interacts with proteins containing phosphorylated tyrosine-X-X-proline (pYXXP) motifs, facilitating complex formation during signaling events. A handful of CRK/CRKL-SH2-specific interactors have been identified to date, although in silico analyses suggest that many additional interactors remain to be found. To identify CRK/CRKL-SH2 interactors with potential involvement in neuronal development, we conducted a mass spectrometry-based proteomics screen using a neuronal cell line (Neuro2A, or N2A). This resulted in the identification of 132 (6 known and 126 novel) YXXP-containing CRK/CRKL-SH2 interactors, of which 77 were stimulated to bind to the CRK/CRKL-SH2 domain following tyrosine phosphatase inhibition. Approximately half of the proteins identified were common interactors of both the CRK- and CRKL-SH2 domains. However, both CRK family member SH2 domains exhibited unique binding partners across experimental replicates. These findings reveal an abundance of novel neuronal CRK/CRKL-SH2 domain binding partners and suggest that CRK family SH2 domains possess undescribed docking preferences beyond the canonical pYXXP motif. KEYWORDS: CRK; CRKL; SH2; LC-MS/MS; Proteomics; Neurodevelopment; Signal Transduction
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Li, Minghua, Zhiqin Li, David L. Morris, and Liangyou Rui. "Identification of SH2B2β as an Inhibitor for SH2B1- and SH2B2α-Promoted Janus Kinase-2 Activation and Insulin Signaling." Endocrinology 148, no. 4 (April 1, 2007): 1615–21. http://dx.doi.org/10.1210/en.2006-1010.
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The SH2B family has three members (SH2B1, SH2B2, and SH2B3) that contain conserved dimerization (DD), pleckstrin homology, and SH2 domains. The DD domain mediates the formation of homo- and heterodimers between members of the SH2B family. The SH2 domain of SH2B1 (previously named SH2-B) or SH2B2 (previously named APS) binds to phosphorylated tyrosines in a variety of tyrosine kinases, including Janus kinase-2 (JAK2) and the insulin receptor, thereby promoting the activation of JAK2 or the insulin receptor, respectively. JAK2 binds to various members of the cytokine receptor family, including receptors for GH and leptin, to mediate cytokine responses. In mice, SH2B1 regulates energy and glucose homeostasis by enhancing leptin and insulin sensitivity. In this work, we identify SH2B2β as a new isoform of SH2B2 (designated as SH2B2α) derived from the SH2B2 gene by alternative mRNA splicing. SH2B2β has a DD and pleckstrin homology domain but lacks a SH2 domain. SH2B2β bound to both SH2B1 and SH2B2α, as demonstrated by both the interaction of glutathione S-transferase-SH2B2β fusion protein with SH2B1 or SH2B2α in vitro and coimmunoprecipitation of SH2B2β with SH2B1 or SH2B2α in intact cells. SH2B2β markedly attenuated the ability of SH2B1 to promote JAK2 activation and subsequent tyrosine phosphorylation of insulin receptor substrate-1 by JAK2. SH2B2β also significantly inhibited SH2B1- or SH2B2α-promoted insulin signaling, including insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1. These data suggest that SH2B2β is an endogenous inhibitor of SH2B1 and/or SH2B2α, negatively regulating insulin signaling and/or JAK2-mediated cellular responses.
6
Songyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X. R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa, and T. Yi. "Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav." Molecular and Cellular Biology 14, no. 4 (April 1994): 2777–85. http://dx.doi.org/10.1128/mcb.14.4.2777-2785.1994.
Full textAbstract:
Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.
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Songyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X. R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa, and T. Yi. "Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav." Molecular and Cellular Biology 14, no. 4 (April 1994): 2777–85. http://dx.doi.org/10.1128/mcb.14.4.2777.
Full textAbstract:
Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.
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Mayer, B. J., and D. Baltimore. "Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase." Molecular and Cellular Biology 14, no. 5 (May 1994): 2883–94. http://dx.doi.org/10.1128/mcb.14.5.2883-2894.1994.
Full textAbstract:
We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.
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Mayer, B. J., and D. Baltimore. "Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase." Molecular and Cellular Biology 14, no. 5 (May 1994): 2883–94. http://dx.doi.org/10.1128/mcb.14.5.2883.
Full textAbstract:
We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.
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Margolis, Ben. "The GRB family of SH2 domain proteins." Progress in Biophysics and Molecular Biology 62, no. 3 (January 1994): 223–44. http://dx.doi.org/10.1016/0079-6107(94)90013-2.
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Wang, Bing, Serge Lemay, Schickwann Tsai, and André Veillette. "SH2 Domain-Mediated Interaction of Inhibitory Protein Tyrosine Kinase Csk with Protein Tyrosine Phosphatase-HSCF." Molecular and Cellular Biology 21, no. 4 (February 15, 2001): 1077–88. http://dx.doi.org/10.1128/mcb.21.4.1077-1088.2001.
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ABSTRACT The protein tyrosine kinase (PTK) Csk is a potent negative regulator of several signal transduction processes, as a consequence of its exquisite ability to inactivate Src-related PTKs. This function requires not only the kinase domain of Csk, but also its Src homology 3 (SH3) and SH2 regions. We showed previously that the Csk SH3 domain mediates highly specific associations with two members of the PEP family of nonreceptor protein tyrosine phosphatases (PTPs), PEP and PTP-PEST. In comparison, the Csk SH2 domain interacts with several tyrosine phosphorylated molecules, presumed to allow targetting of Csk to sites of Src family kinase activation. Herein, we attempted to understand better the regulation of Csk by identifying ligands for its SH2 domain. Using a modified yeast two-hybrid screen, we uncovered the fact that Csk associates with PTP-HSCF, the third member of the PEP family of PTPs. This association was documented not only in yeast cells but also in a heterologous mammalian cell system and in cytokine-dependent hemopoietic cells. Surprisingly, the Csk–PTP-HSCF interaction was found to be mediated by the Csk SH2 domain and two putative sites of tyrosine phosphorylation in the noncatalytic portion of PTP-HSCF. Transfection experiments indicated that Csk and PTP-HSCF synergized to inhibit signal transduction by Src family kinases and that this cooperativity was dependent on the domains mediating their association. Finally, we obtained evidence that PTP-HSCF inactivated Src-related PTKs by selectively dephosphorylating the positive regulatory tyrosine in their kinase domain. Taken together, these results demonstrate that part of the function of the Csk SH2 domain is to mediate an inducible association with a PTP, thereby engineering a more efficient inhibitory mechanism for Src-related PTKs. Coupled with previously published observations, these data also establish that Csk forms complexes with all three known members of the PEP family.
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Kurzer, Jason H., Pipsa Saharinen, Olli Silvennoinen, and Christin Carter-Su. "Binding of SH2-B Family Members within a Potential Negative Regulatory Region Maintains JAK2 in an Active State." Molecular and Cellular Biology 26, no. 17 (September 1, 2006): 6381–94. http://dx.doi.org/10.1128/mcb.00570-06.
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ABSTRACT The tyrosine kinase Janus kinase 2 (JAK2) transduces signaling for the majority of known cytokine receptor family members and is constitutively activated in some cancers. Here we examine the mechanisms by which the adapter proteins SH2-Bβ and APS regulate the activity of JAK2. We show that like SH2-Bβ, APS binds JAK2 at multiple sites and that binding to phosphotyrosine 813 is essential for APS to increase active JAK2 and to be phosphorylated by JAK2. Binding of APS to a phosphotyrosine 813-independent site inhibits JAK2. Both APS and SH2-Bβ increase JAK2 activity independent of their N-terminal dimerization domains. SH2-Bβ-induced increases in JAK2 dimerization require only the SH2 domain and only one SH2-Bβ to be bound to a JAK2 dimer. JAK2 mutations and truncations revealed that amino acids 809 to 811 in JAK2 are a critical component of a larger regulatory region within JAK2, most likely including amino acids within the JAK homology 1 (JH1) and JH2 domains and possibly the FERM domain. Together, our data suggest that SH2-Bβ and APS do not activate JAK2 as a consequence of their own dimerization, recruitment of an activator of JAK2, or direct competition with a JAK2 inhibitor for binding to JAK2. Rather, they most likely induce or stabilize an active conformation of JAK2.
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Matthews, R. J., D. B. Bowne, E. Flores, and M. L. Thomas. "Characterization of hematopoietic intracellular protein tyrosine phosphatases: description of a phosphatase containing an SH2 domain and another enriched in proline-, glutamic acid-, serine-, and threonine-rich sequences." Molecular and Cellular Biology 12, no. 5 (May 1992): 2396–405. http://dx.doi.org/10.1128/mcb.12.5.2396-2405.1992.
Full textAbstract:
Protein tyrosine phosphatases (PTPases) are a family of enzymes important in cellular regulation. Characterization of two cDNAs encoding intracellular PTPases expressed primarily in hematopoietic tissues and cell lines has revealed proteins that are potential regulators of signal transduction. One of these, SHP (Src homology region 2 [SH2]-domain phosphatase), possesses two tandem SH2 domains at the amino terminus of the molecule. SH2 domains have previously been described in proteins implicated in signal transduction, and SHP may be one of a family of nonreceptor PTPases that can act as direct antagonists to the nonreceptor protein tyrosine kinases. The SH2 domains of SHP preferentially bind a 15,000-Mr protein expressed by LSTRA cells. LSTRA cells were shown to express SHP protein by immunoprecipitation, thus demonstrating a potential physiological interaction. The other PTPase, PEP (proline-, glutamic acid-, serine-, and threonine-rich [PEST]-domain phosphatase), is distinguished by virtue of a large carboxy-terminal domain of approximately 500 amino acids that is rich in PEST residues. PEST sequences are found in proteins that are rapidly degraded. Both proteins have been expressed by in vitro transcription and translation and in bacterial expression systems, and both have been demonstrated to have PTPase activity. These two additional members of the PTPase family accentuate the variety of PTPase structures and indicate the potential diversity of function for intracellular tyrosine phosphatases.
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Matthews, R. J., D. B. Bowne, E. Flores, and M. L. Thomas. "Characterization of hematopoietic intracellular protein tyrosine phosphatases: description of a phosphatase containing an SH2 domain and another enriched in proline-, glutamic acid-, serine-, and threonine-rich sequences." Molecular and Cellular Biology 12, no. 5 (May 1992): 2396–405. http://dx.doi.org/10.1128/mcb.12.5.2396.
Full textAbstract:
Protein tyrosine phosphatases (PTPases) are a family of enzymes important in cellular regulation. Characterization of two cDNAs encoding intracellular PTPases expressed primarily in hematopoietic tissues and cell lines has revealed proteins that are potential regulators of signal transduction. One of these, SHP (Src homology region 2 [SH2]-domain phosphatase), possesses two tandem SH2 domains at the amino terminus of the molecule. SH2 domains have previously been described in proteins implicated in signal transduction, and SHP may be one of a family of nonreceptor PTPases that can act as direct antagonists to the nonreceptor protein tyrosine kinases. The SH2 domains of SHP preferentially bind a 15,000-Mr protein expressed by LSTRA cells. LSTRA cells were shown to express SHP protein by immunoprecipitation, thus demonstrating a potential physiological interaction. The other PTPase, PEP (proline-, glutamic acid-, serine-, and threonine-rich [PEST]-domain phosphatase), is distinguished by virtue of a large carboxy-terminal domain of approximately 500 amino acids that is rich in PEST residues. PEST sequences are found in proteins that are rapidly degraded. Both proteins have been expressed by in vitro transcription and translation and in bacterial expression systems, and both have been demonstrated to have PTPase activity. These two additional members of the PTPase family accentuate the variety of PTPase structures and indicate the potential diversity of function for intracellular tyrosine phosphatases.
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Berg, Angela, Martin Gräber, Sebastian Schmutzler, Ralf Hoffmann, and Thorsten Berg. "A High-Throughput Fluorescence Polarization-Based Assay for the SH2 Domain of STAT4." Methods and Protocols 5, no. 6 (November 23, 2022): 93. http://dx.doi.org/10.3390/mps5060093.
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The signal transducer and activation of transcription (STAT) proteins are a family of Src homology 2 (SH2) domain-containing transcription factors. The family member STAT4 is a mediator of IL-12 signalling and has been implicated in the pathogenesis of multiple autoimmune diseases. The activity of STAT4 requires binding of phosphotyrosine-containing motifs to its SH2 domain. Selective inhibitors of the STAT4 SH2 domain have not been published to date. Here, we present a fluorescence polarization-based assay for the identification of inhibitors of the STAT4 SH2 domain. The assay is based on the interaction between the STAT4 SH2 domain and the fluorophore-labelled peptide 5-carboxyfluorescein-GpYLPQNID (Kd = 34 ± 4 nM). The assay is stable with respect to DMSO concentrations of up to 10% and incubation times of at least 8 h. The Z’-value of 0.85 ± 0.01 indicates that the assay is suited for use in high-throughput screening campaigns aimed at identifying new therapeutic modalities for the treatment of autoimmune diseases.
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Patsoukis, Nikolaos, Jonathan Duke-Cohan, Apoorvi Chaudhri, Eunyoung Park, Council Asia, Anders Berg, Gordon J. Freeman, Michael Eck, and Vassiliki A. Boussiotis. "The Two SH2 Domains of SHP-2 Bridge Two PD-1 Molecules Resulting in SHP-2 Activation and PD-1-Mediated Inhibition." Blood 132, Supplement 1 (November 29, 2018): 862. http://dx.doi.org/10.1182/blood-2018-99-117486.
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Abstract Programmed cell death-1 (PD-1) is a checkpoint inhibitory receptor member of the B7-CD28 family, which promotes peripheral tolerance and restrains anti-viral and anti-tumor immunity. Although PD-1 blockade leads to durable clinical responses in a fraction of patients, the majority of patients display only transient responses, emphasizing the need for better understanding of the mechanism of PD-1-mediated T cell inhibition. In contrast to other CD28 family family members, which form disulfide-linked homodimers, PD-1 lacks the juxtamembrane cysteine residue responsible for homodimer formation and has been shown to exist as a monomer. The cytoplasmic tail of PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM), centered on Y223 residue, and one immunoreceptor tyrosine-based switch motif (ITSM), centered on Y248 residue. Mutational studies have shown that PD-1-mediated inhibition relies on the interaction of the ITSM with SHP-2 but the mechanism by which PD-1 induces SHP-2 activation is unknown. In this study we sought to determine how PD-1: SHP-2 interaction leads to inhibition of T-cell responses. SHP-2 contains a phosphatase (PTP) domain and two SH2 domains, N-SH2 and C-SH2. To determine whether PD-1 selectively interacts with a specific SH2 domain of SHP-2, we generated five different GST-fusion proteins in which GST was fused with either SHP-2 full length (FL), SHP-2-N-SH2, SHP-2-C-SH2, SHP-2-ΔN-SH2 (lacking the N-terminus SH2 domain) or SHP-2-PTP. Surprisingly, pull-down assays with GST-fusion proteins using lysates from primary human T cells or Jurkat T cell line revealed that PD-1 interacted with SHP-2 via both SH2 domains of SHP-2. Biacore assays confirmed that phosphorylated ITSM Y248 interacted with SHP-2 N-SH2 and C-SH2. To determine whether one of the SHP-2 SH2 domains might preferentially interact with PD-1 in live cells, we mutagenized the functional sites of the N-SH2 or C-SH2 domain at arginine R32 and R138, respectively, and transfected COS cells with cDNA of SHP-2 wild type or each SHP-2 SH2 mutant, together with PD-1 and the TCR proximal kinase Fyn, required for PD-1 phosphorylation and interaction with SHP-2. Immunoprecipitation and immunoblot assays showed that mutagenesis of either SH2 domain abrogated interaction of SHP-2 with PD-1 ITSM Y248, providing evidence that both SH2 domains of SHP-2 are involved in the interaction with PD-1. Because each PD-1 molecule has only one ITSM, these results indicate that SHP-2 interacts via its two SH2 domains with two PD-1 molecules. Assessment of PD-1: PD-1 dimer formation in live cells by split luciferase complementation, using NanoBiT proximity assays, showed that upon PD-1 phosphorylation, PD-1: PD1 interaction occurs only in the presence of SHP-2 with intact N-SH2 and C-SH2 domains. The SH2 domains of SHP-2 have a crucial and distinct role in regulating SHP-2 PTPase activity. In the absence of a tyrosine-phosphorylated binding ligand, N-SH2 is bound to the PTP domain leading to an auto-inhibitory closed conformation that blocks the PTP active site. Phosphorylation of Y542 in the SHP-2 C-terminus tail leads to intramolecular interaction of Y542 with the N-SH2 domain to relieve binding to PTP domain. Similarly, intermolecular interaction of the N-SH2 domain with specific phosphorylated ligands disrupts its PTP recognition surface reversing the auto-inhibitory conformation and activates the phosphatase. We determined that monomeric PD-1 ITIM-pY223 or ITSM-pY248 peptides did not activate the PTP. A dimeric phosphopeptide generated by covalent joining of two PD-1 ITSM-pY248 phosphopeptides with a linker that matches the distance between the binding sites of the two SHP-2 SH2 domains induced rapid activation of SHP-2. In contrast, similarly designed dimeric peptides joining either two PD-1 ITIM-pY223 or ITIM-pY223-and-ITSM-pY248 phosphopeptides did not activate PTP activity of SHP-2. The ability of PD1 ITSM-pY248 to induce SHP-2 activation was correlated with inhibition of antigen-mediated IL-2 production, which was abrogated when Y248 was mutagenized to phenylalanine. Our results reveal the geometry of PD-1: SHP-2 interaction that leads to SHP-2 activation and have implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or enhance T cell responses by disrupting PD-1 dimerization and SHP-2 activation. Disclosures Freeman: EMD-Serono: Patents & Royalties; Xios: Membership on an entity's Board of Directors or advisory committees; Origimed: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Patents & Royalties; Bristol-Myers-Squibb: Patents & Royalties; Bristol-Myers-Squibb: Membership on an entity's Board of Directors or advisory committees; Roche: Patents & Royalties; Dako: Patents & Royalties; Boehringer-Ingelheim: Patents & Royalties; Merck: Patents & Royalties; Novartis: Patents & Royalties.
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Shen, Kexin, Jamie A. Moroco, Ravi K. Patel, Haibin Shi, John R. Engen, Heather R. Dorman, and Thomas E. Smithgall. "The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation." Science Signaling 11, no. 553 (October 23, 2018): eaat5916. http://dx.doi.org/10.1126/scisignal.aat5916.
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Fgr is a member of the Src family of nonreceptor tyrosine kinases, which are overexpressed and constitutively active in many human cancers. Fgr expression is restricted to myeloid hematopoietic cells and is markedly increased in a subset of bone marrow samples from patients with acute myeloid leukemia (AML). Here, we investigated the oncogenic potential of Fgr using Rat-2 fibroblasts that do not express the kinase. Expression of either wild-type or regulatory tail-mutant constructs of Fgr promoted cellular transformation (inferred from colony formation in soft agar), which was accompanied by phosphorylation of the Fgr activation loop, suggesting that the kinase domain of Fgr functions independently of regulation by its noncatalytic SH3-SH2 region. Unlike other family members, recombinant Fgr was not activated by SH3-SH2 domain ligands. However, hydrogen-deuterium exchange mass spectrometry data suggested that the regulatory SH3 and SH2 domains packed against the back of the kinase domain in a Src-like manner. Sequence alignment showed that the activation loop of Fgr was distinct from that of all other Src family members, with proline rather than alanine at the +2 position relative to the activation loop tyrosine. Substitution of the activation loop of Fgr with the sequence from Src partially inhibited kinase activity and suppressed colony formation. Last, Fgr expression enhanced the sensitivity of human myeloid progenitor cells to the cytokine GM-CSF. Because its kinase domain is not sensitive to SH3-SH2–mediated control, simple overexpression of Fgr without mutation may contribute to oncogenic transformation in AML and other blood cancers.
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Wang, Lawrence L., Julie Blasioli, David R. Plas, Matthew L. Thomas, and Wayne M. Yokoyama. "Specificity of the SH2 Domains of SHP-1 in the Interaction with the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing Receptor gp49B." Journal of Immunology 162, no. 3 (February 1, 1999): 1318–23. http://dx.doi.org/10.4049/jimmunol.162.3.1318.
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Abstract Inhibitory receptors on hemopoietic cells critically regulate cellular function. Despite their expression on a variety of cell types, these inhibitory receptors signal through a common mechanism involving tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM), which engages Src homology 2 (SH2) domain-containing cytoplasmic tyrosine or inositol phosphatases. In this study, we have investigated the proximal signal-transduction pathway of an ITIM-bearing receptor, gp49B, a member of a newly described family of murine NK and mast cell receptors. We demonstrate that the tyrosine residues within the ITIMs are phosphorylated and serve for the association and activation of the cytoplasmic tyrosine phosphatase SHP-1. Furthermore, we demonstrate a physiologic association between gp49B and SHP-1 by coimmunoprecipitation studies from NK cells. To address the mechanism of binding between gp49B and SHP-1, binding studies involving glutathione S-transferase SHP-1 mutants were performed. Utilizing the tandem SH2 domains of SHP-1, we show that either SH2 domain can interact with phosphorylated gp49B. Full-length SHP-1, with an inactivated amino SH2 domain, also retained gp49B binding. However, binding to gp49B was disrupted by inactivation of the carboxyl SH2 domain of full-length SHP-1, suggesting that in the presence of the phosphatase domain, the carboxyl SH2 domain is required for the recruitment of phosphorylated gp49B. Thus, gp49B signaling involves SHP-1, and this association is dependent on tyrosine phosphorylation of the gp49B ITIMs, and an intact SHP-1 carboxyl SH2 domain.
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Mayoral-Varo, Víctor, María Pilar Sánchez-Bailón, Annarica Calcabrini, Marta García-Hernández, Valerio Frezza, María Elena Martín, Víctor M. González, and Jorge Martín-Pérez. "The Relevance of the SH2 Domain for c-Src Functionality in Triple-Negative Breast Cancer Cells." Cancers 13, no. 3 (January 26, 2021): 462. http://dx.doi.org/10.3390/cancers13030462.
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The role of Src family kinases (SFKs) in human tumors has been always associated with tyrosine kinase activity and much less attention has been given to the SH2 and SH3 adapter domains. Here, we studied the role of the c-Src-SH2 domain in triple-negative breast cancer (TNBC). To this end, SUM159PT and MDA-MB-231 human cell lines were employed as model systems. These cells conditionally expressed, under tetracycline control (Tet-On system), a c-Src variant with point-inactivating mutation of the SH2 adapter domain (R175L). The expression of this mutant reduced the self-renewal capability of the enriched population of breast cancer stem cells (BCSCs), demonstrating the importance of the SH2 adapter domain of c-Src in the mammary gland carcinogenesis. In addition, the analysis of anchorage-independent growth, proliferation, migration, and invasiveness, all processes associated with tumorigenesis, showed that the SH2 domain of c-Src plays a very relevant role in their regulation. Furthermore, the transfection of two different aptamers directed to SH2-c-Src in both SUM159PT and MDA-MB-231 cells induced inhibition of their proliferation, migration, and invasiveness, strengthening the hypothesis that this domain is highly involved in TNBC tumorigenesis. Therefore, the SH2 domain of c-Src could be a promising therapeutic target and combined treatments with inhibitors of c-Src kinase enzymatic activity may represent a new therapeutic strategy for patients with TNBC, whose prognosis is currently very negative.
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Weerawarna, Pathum M., and Timothy I. Richardson. "Lyn Kinase Structure, Regulation, and Involvement in Neurodegenerative Diseases: A Mini Review." Kinases and Phosphatases 1, no. 1 (January 23, 2023): 23–38. http://dx.doi.org/10.3390/kinasesphosphatases1010004.
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LYN proto-oncogene, Src family tyrosine kinase (Lyn) is a tyrosine kinase that belongs to the Src family (SFK). It is expressed as two isoforms in humans, LynA and LynB. Like other SFKs, Lyn consists of five protein domains, an N-terminal SH4 domain followed by a unique domain, the SH3 and SH2 domains, and a catalytic SH1 domain. The autophosphorylation of Tyr397 activates the protein, while the phosphorylation of the C-terminal inhibitory Tyr508 by C-terminal Src kinase (Csk) or Csk homologous kinase (Chk) inhibits the catalytic activity. The interaction of the SH2 domain with the phosphorylated Tyr508 stabilizes a compact, self-inhibited state. The interaction of the SH3 domain with a linker between the SH2 and catalytic domains further stabilizes this inactive conformation. The two critical structural features of the catalytic domain are a conserved DFG moiety and the αC helix, which can adopt in or out conformations. In the active state, both the DFG moiety and αC helix adopt in conformations, while in the inactive state, they adopt out conformations. Lyn has well-established functions in various hematopoietic cell types and more recent studies have revealed its roles in non-hematopoietic cells. At the molecular level, these functions are mainly exerted by phosphorylating specific tyrosine residues in immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activator motifs (ITAMs) associated with cell surface receptors. The phosphorylation of ITAMs by Lyn can initiate either activating or inhibitory (ITAMi) cell signaling depending on the receptor, targeting mode (crosslinking or monovalent targeting), and the cellular context. The phosphorylation of ITIMs by Lyn initiates inhibitory cell signaling via the recruitment of phosphatases to the ITIM-bearing receptor. The role of Lyn in cancer and autoimmune diseases has been extensively discussed in the literature. The involvement of Lyn in neurodegenerative diseases has been described more recently and, as such, it is now an emerging target for the treatment of neurodegenerative diseases.
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Sheets, Michael P., Usha P. Warrior, Hosup Yoon, Karl W. Mollison, Stevan W. Djuric, and James M. Trevillyan. "A High-Capacity Scintillation Proximity Assay for the Discovery and Evaluation of ZAP-70 Tandem SH2 Domain Antagonists." Journal of Biomolecular Screening 3, no. 2 (March 1998): 139–44. http://dx.doi.org/10.1177/108705719800300208.
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A scintillation proximity assay (SPA) is described, which quantitates the ability of compounds to inhibit the binding interaction of a select phosphopeptide with the tandem SH2 domains of the ZAP-70 protein tyrosine kinase. The method is based on the ability of a truncated ZAP-70 tandem SH2 domain-derived peptide to bind an 125I-labeled, diphosphorylated peptide corresponding to the human T-cell receptor ζ-1 immunoglobulin receptor family tyrosine-based activation motif (ITAM). ZAP-70 tandem SH2 domain peptide was biotinylated and bound to streptavidin-coated SPA beads. 125I-labeled ζ-1 ITAM ([125I]-ζ-1 ITAM) bound to immobilized ZAP-70 tandem SH2 domain peptide in a saturable, time- and peptide concentration-dependent fashion. Unlabeled diphosphorylated ζ-1 ITAM competed binding with an ICso value equal to approximately 10-15 nM. Binding of ζ-1 ITAM to the ZAP-70 tandem SH2 domain was dependent on the cooperative interaction of the dual phosphotyrosine residues. Unlabeled monotyrosyl-phosphorylated peptides failed to compete with [125I]-ζ-1 ITAM binding to ZAP-70 SH2 domain. Also, labeled monotyrosyl-phosphorylated peptides failed to associate with the ZAP-70 SH2 domain in direct binding studies. Association and dissociation binding kinetics were determined to be extremely rapid at room temperature, reaching equilibrium within 5 min. The Kd for [125I]-ζ-1 ITAM binding to ZAP-70 tandem SH2 domain peptide was determined by Scatchard analysis to be 1.5-2 nM. The SPA assay was adapted for automated, high-capacity screening, which allowed evaluation of 23,040 small molecular weight compounds per day. The assay is useful for both drug discovery and as a research tool for the study of binding interactions between signal-transducing molecules critical for T-cell activation.
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Kan, Yagmur, YiTing Paung, Markus A. Seeliger, and W. Todd Miller. "Domain Architecture of the Nonreceptor Tyrosine Kinase Ack1." Cells 12, no. 6 (March 15, 2023): 900. http://dx.doi.org/10.3390/cells12060900.
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The nonreceptor tyrosine kinase (NRTK) Ack1 comprises a distinct arrangement of non-catalytic modules. Its SH3 domain has a C-terminal to the kinase domain (SH1), in contrast to the typical SH3-SH2-SH1 layout in NRTKs. The Ack1 is the only protein that shares a region of high homology to the tumor suppressor protein Mig6, a modulator of EGFR. The vertebrate Acks make up the only tyrosine kinase (TK) family known to carry a UBA domain. The GTPase binding and SAM domains are also uncommon in the NRTKs. In addition to being a downstream effector of receptor tyrosine kinases (RTKs) and integrins, Ack1 can act as an epigenetic regulator, modulate the degradation of the epidermal growth factor receptor (EGFR), confer drug resistance, and mediate the progression of hormone-sensitive tumors. In this review, we discuss the domain architecture of Ack1 in relation to other protein kinases that possess such defined regulatory domains.
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AHMED, Zamal, Beverley J. SMITH, Kei KOTANI, Peter WILDEN, and Tahir S. PILLAY. "APS, an adapter protein with a PH and SH2 domain, is a substrate for the insulin receptor kinase." Biochemical Journal 341, no. 3 (July 26, 1999): 665–68. http://dx.doi.org/10.1042/bj3410665.
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APS (adapter protein with a PH and SH2 domain) is the newest member of a family of tyrosine kinase adapter proteins including SH2-B and Lnk. We previously identified SH2-B as an insulin-receptor-binding protein and substrate [Kotani, Wilden and Pillay (1998) Biochem J. 335, 103-109]. Here we show that APS interacts with the insulin receptor kinase activation loop through its SH2 domain and insulin stimulates the tyrosine-phosphorylation of APS. Furthermore, the phosphorylation of activation-loop tyrosine residues 1158 and 1162 are required for this interaction.
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Ge, Liang, Bo Wu, Youjia Zhang, Jiarong Wang, Hongxin Zhao, and Junfeng Wang. "Biochemical and NMR characterization of the interactions of Vav2–SH2 domain with lipids and the EphA2 juxtamembrane region on membrane." Biochemical Journal 477, no. 19 (October 12, 2020): 3791–801. http://dx.doi.org/10.1042/bcj20200300.
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Vav2 is a ubiquitous guanine nucleotide exchange factor (GEF) for Rho family GTPases that is involved in regulating a wide range of biological processes. It interacts with several tyrosine-phosphorylated cell surface receptors, including the Eph family receptors, through its SH2 domain. The interaction of Vav2 with EphA2 is crucial for EphA2-mediated tumor angiogenesis. Here we show that Vav2–SH2 domain is a lipid-binding module that can recognize PI(4,5)P2 and PI(3,4,5)P3 lipids weakly but specifically. The specific lipid-binding site in Vav2–SH2 domain was identified by NMR chemical shift perturbation experiments using the head groups of PI(4,5)P2 and PI(3,4,5)P3, both of which bind to Vav2–SH2 with millimolar binding affinities. In addition, the interaction between Vav2–SH2 and the phosphorylated juxtamembrane region (JM) of EphA2 (Y594 phosphorylated) was investigated using NMR techniques. Furthermore, by using a nickel–lipid containing peptide-based nanodiscs system, we studied the binding of Vav2–SH2 to the phosphorylated JM region of EphA2 on lipid membrane and uncovered a role of membrane environment in modulating this protein–protein recognition.
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Duplay, P., M. Thome, F. Hervé, and O. Acuto. "p56lck interacts via its src homology 2 domain with the ZAP-70 kinase." Journal of Experimental Medicine 179, no. 4 (April 1, 1994): 1163–72. http://dx.doi.org/10.1084/jem.179.4.1163.
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p56lck, a member of the src family of protein tyrosine kinases, is an essential component in T cell receptor (TCR) signal transduction. p56lck contains a src homology 2 (SH2) domain found in a number of proteins involved in intracellular signaling. SH2 domains have been implicated in protein-protein interactions by binding to sequences in target proteins containing phosphorylated tyrosine. Using an in vitro assay, we have studied specific binding of tyrosine-phosphorylated proteins to a recombinant p56lck SH2 domain. In nonactivated Jurkat cells, two tyrosine-phosphorylated proteins were detected. Stimulation with anti-CD3 monoclonal antibodies induced the binding of seven additional tyrosine-phosphorylated proteins to the SH2 domain of p56lck. We have identified the zeta-associated tyrosine kinase, ZAP-70, as one of these proteins. Evidence suggests that binding of ZAP-70 to p56lck SH2 is direct and not mediated by zeta. The significance of this interaction was further investigated in vivo. p56lck could be coprecipitated with the zeta/ZAP-70 complex and conversely, ZAP-70 was detected in p56lck immunoprecipitates of activated Jurkat cells. The physical association of p56lck and ZAP-70 during activation supports the recently proposed functional cooperation of these two tyrosine kinases in TCR signaling.
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Ahmed, Z., and T. S. Pillay. "Functional effects of APS and SH2-B on insulin receptor signalling." Biochemical Society Transactions 29, no. 4 (August 1, 2001): 529–34. http://dx.doi.org/10.1042/bst0290529.
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APS [for ‘adapter protein with a pleckstrin homology (PH) and Src homology 2 (SH2) domain’] belongs to a family of adapter proteins involved in signalling by the receptors for insulin, insulin-like growth factor 1, platelet-derived growth factor and nerve growth factor. Other members include alternatively spliced SH2-B isoforms (SH2Bα. SH2-Byβ and SH2-Bγ) and Lnk. These have a C-terminal SH2 domain, a central PH domain and an N-terminal proline-rich region. SH2Bα, APS and Lnk have a conserved C-terminal tyrosine phosphorylation site, whereas the alternatively spliced SH2-Bβ and SH2-Bγ have distinct C-termini. There is considerable sequence similarity between APS, SH2-B and Lnk, particularly in the SH2 domain. Both APS and SH2-Bα interact with the insulin-receptor activation loop phosphorylation sites and undergo insulin-stimulated tyrosine phosphorylation, although the phosphorylation of SH2-B is considerably weaker. APS couples c-Cbl to the insulin receptor, resulting in ubiquitination of the insulin receptor. We established cell lines [Chinese hamster ovary (CHO). T-APS and CHO. T-SH2-B cells] overexpressing APS and SH2-Bα to study their roles in insulin receptor signalling. Either adapter protein enhances insulin receptor and ERK (extracellular-signal-regulated kinase) phosphorylation. In CHO. T-APS cells, Akt phosphorylation is observed earlier than in CHO.T-SH2-B cells. Both enhance insulin-stimulated Akt activation but APS seems to cause greater activation. Thus APS and SH2-B have similar effects on insulin receptor signalling, although the effects of SH2-B are independent of its phosphorylation.
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Asada, Hiroshi, Naoto Ishii, Yoshiteru Sasaki, Kazuhiro Endo, Hirotake Kasai, Nobuyuki Tanaka, Toshikazu Takeshita, Shigeru Tsuchiya, Tasuke Konno, and Kazuo Sugamura. "Grf40, A Novel Grb2 Family Member, Is Involved in T Cell Signaling through Interaction with SLP-76 and LAT." Journal of Experimental Medicine 189, no. 9 (May 3, 1999): 1383–90. http://dx.doi.org/10.1084/jem.189.9.1383.
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We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain–containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76–dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.
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Nishi, Masahiro, Eric D. Werner, Byung-Chul Oh, J. Daniel Frantz, Sirano Dhe-Paganon, Lone Hansen, Jongsoon Lee, and Steven E. Shoelson. "Kinase Activation through Dimerization by Human SH2-B." Molecular and Cellular Biology 25, no. 7 (April 1, 2005): 2607–21. http://dx.doi.org/10.1128/mcb.25.7.2607-2621.2005.
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ABSTRACT The isoforms of SH2-B, APS, and Lnk form a family of signaling proteins that have been described as activators, mediators, or inhibitors of cytokine and growth factor signaling. We now show that the three alternatively spliced isoforms of human SH2-B readily homodimerize in yeast two-hybrid and cellular transfections assays, and this is mediated specifically by a unique domain in its amino terminus. Consistent with previous reports, we further show that the SH2 domains of SH2-B and APS bind JAK2 at Tyr813. These findings suggested a model in which two molecules of SH2-B or APS homodimerize with their SH2 domains bound to two JAK2 molecules, creating heterotetrameric JAK2-(SH2-B)2-JAK2 or JAK2-(APS)2-JAK2 complexes. We further show that APS and SH2-B isoforms heterodimerize. At lower levels of SH2-B or APS expression, dimerization approximates two JAK2 molecules to induce transactivation. At higher relative concentrations of SH2-B or APS, kinase activation is blocked. SH2-B or APS homodimerization and SH2-B/APS heterodimerization thus provide direct mechanisms for activating and inhibiting JAK2 and other kinases from the inside of the cell and for potentiating or attenuating cytokine and growth factor receptor signaling when ligands are present.
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Mehlmann, Lisa M., and Laurinda A. Jaffe. "SH2 domain-mediated activation of an SRC family kinase is not required to initiate Ca2+ release at fertilization in mouse eggs." Reproduction 129, no. 5 (May 2005): 557–64. http://dx.doi.org/10.1530/rep.1.00638.
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SRC family kinases (SFKs) function in initiating Ca2+release at fertilization in several species in the vertebrate evolutionary line, but whether they play a similar role in mammalian fertilization has been uncertain. We investigated this question by first determining which SFK proteins are expressed in mouse eggs, and then measuring Ca2+release at fertilization in the presence of dominant negative inhibitors. FYN and YES proteins were found in mouse eggs, but other SFKs were not detected; based on this, we injected mouse eggs with a mixture of FYN and YES Src homology 2 (SH2) domains. These SH2 domains were effective inhibitors of Ca2+release at fertilization in starfish eggs, but did not inhibit Ca2+release at fertilization in mouse eggs. Thus the mechanism by which sperm initiate Ca2+release in mouse eggs does not depend on SH2 domain-mediated activation of an SFK. We also tested the small molecule SFK inhibitor SU6656, and found that it became compartmentalized in the egg cytoplasm, thus suggesting caution in the use of this inhibitor. Our findings indicate that although the initiation of Ca2+release at fertilization of mammalian eggs occurs by a pathway that has many similarities to that in evolutionarily earlier animal groups, the requirement for SH2 domain-mediated activation of an SFK is not conserved.
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Jiao, H., K. Berrada, W. Yang, M. Tabrizi, L. C. Platanias, and T. Yi. "Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1." Molecular and Cellular Biology 16, no. 12 (December 1996): 6985–92. http://dx.doi.org/10.1128/mcb.16.12.6985.
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SHP-1 is an SH2-containing cytoplasmic tyrosine phosphatase that is widely distributed in cells of the hematopoietic system. SHP-1 plays an important role in the signal transduction of many cytokine receptors, including the receptor for erythropoietin, by associating via its SH2 domains to the receptors and dephosphorylating key substrates. Recent studies have suggested that SHP-1 regulates the function of Jak family tyrosine kinases, as shown by its constitutive association with the Tyk2 kinase and the hyperphosphorylation of Jak kinases in the motheaten cells that lack functional SHP-1. We have examined the interactions of SHP-1 with two tyrosine kinases activated during engagement of the erythropoietin receptor, the Janus family kinase Jak-2 and the c-fps/fes kinase. Immunoblotting studies with extracts from mouse hematopoietic cells demonstrated that Jak2, but not c-fes, was present in anti-SHP-1 immunoprecipitates, suggesting that SHP-1 selectively associates with Jak2 in vivo. Consistent with this, when SHP-1 was coexpressed with these kinases in Cos-7 cells, it associated with and dephosphorylated Jak2 but not c-fes. Transient cotransfection of truncated forms of SHP-1 with Jak2 demonstrated that the SHP-1-Jak2 interaction is direct and is mediated by a novel binding activity present in the N terminus of SHP-1, independently of SH2 domain-phosphotyrosine interaction. Such SHP-1-Jak2 interaction resulted in induction of the enzymatic activity of the phosphatase in in vitro protein tyrosine phosphatase assays. Interestingly, association of the SH2n domain of SHP-1 with the tyrosine phosphorylated erythropoietin receptor modestly potentiated but was not essential for SHP-1-mediated dephosphorylation of Jak2 and had no effect on c-fes phosphorylation. These data indicate that the main mechanism for regulation of Jak2 phosphorylation by SHP-1 involves a direct, SH2-independent interaction with Jak2 and suggest the existence of similar mechanisms for other members of the Jak family of kinases. They also suggest that such interactions may provide one of the mechanisms that control SHP-1 substrate specificity.
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Kohmura, N., T. Yagi, Y. Tomooka, M. Oyanagi, R. Kominami, N. Takeda, J. Chiba, Y. Ikawa, and S. Aizawa. "A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption." Molecular and Cellular Biology 14, no. 10 (October 1994): 6915–25. http://dx.doi.org/10.1128/mcb.14.10.6915-6925.1994.
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We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function.
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Kohmura, N., T. Yagi, Y. Tomooka, M. Oyanagi, R. Kominami, N. Takeda, J. Chiba, Y. Ikawa, and S. Aizawa. "A novel nonreceptor tyrosine kinase, Srm: cloning and targeted disruption." Molecular and Cellular Biology 14, no. 10 (October 1994): 6915–25. http://dx.doi.org/10.1128/mcb.14.10.6915.
Full textAbstract:
We have isolated a novel nonreceptor tyrosine kinase, Srm, that maps to the distal end of chromosome 2. It has SH2, SH2', and SH3 domains and a tyrosine residue for autophosphorylation in the kinase domain but lacks an N-terminal glycine for myristylation and a C-terminal tyrosine which, when phosphorylated, suppresses kinase activity. These are structural features of the recently identified Tec family of nonreceptor tyrosine kinases. The Srm N-terminal unique domain, however, lacks the structural characteristics of the Tec family kinases, and the sequence similarity is highest to Src in the SH region. The expression of two transcripts is rather ubiquitous and changes according to tissue and developmental stage. Mutant mice were generated by gene targeting in embryonic stem cells but displayed no apparent phenotype as in mutant mice expressing Src family kinases. These results suggest that Srm constitutes a new family of nonreceptor tyrosine kinases that may be redundant in function.
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Cobb, B. S., M. D. Schaller, T. H. Leu, and J. T. Parsons. "Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK." Molecular and Cellular Biology 14, no. 1 (January 1994): 147–55. http://dx.doi.org/10.1128/mcb.14.1.147-155.1994.
Full textAbstract:
Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.
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Cobb, B. S., M. D. Schaller, T. H. Leu, and J. T. Parsons. "Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK." Molecular and Cellular Biology 14, no. 1 (January 1994): 147–55. http://dx.doi.org/10.1128/mcb.14.1.147.
Full textAbstract:
Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.
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Chen, Riyan, Sylvain Latour, Xiaochu Shi, and André Veillette. "Association between SAP and FynT: Inducible SH3 Domain-Mediated Interaction Controlled by Engagement of the SLAM Receptor." Molecular and Cellular Biology 26, no. 15 (August 1, 2006): 5559–68. http://dx.doi.org/10.1128/mcb.00357-06.
Full textAbstract:
ABSTRACT SAP is an intracellular adaptor molecule composed almost exclusively of an SH2 domain. It is mutated in patients with X-linked lymphoproliferative disease, a human immunodeficiency. Several immune abnormalities were also identified in SAP-deficient mice. By way of its SH2 domain, SAP interacts with tyrosine-based motifs in the cytoplasmic domain of SLAM family receptors. SAP promotes SLAM family receptor-induced protein tyrosine phosphorylation, due to its capacity to recruit the Src-related kinase FynT. This unusual property relies on the existence of a second binding surface in the SAP SH2 domain, centered on arginine 78 of SAP, that binds directly to the FynT SH3 domain. Herein, we wanted to further understand the mechanisms controlling the interaction between SLAM-SAP and FynT. Our experiments showed that, unlike conventional associations mediated by SH3 domains, the interaction of the FynT SH3 domain with SLAM-SAP was strictly inducible. It was absolutely dependent on engagement of SLAM by extracellular ligands. We obtained evidence that this inducibility was not due to increased binding of SLAM to SAP following SLAM engagement. Furthermore, it could occur independently of any appreciable SLAM-dependent biochemical signal. In fact, our data indicated that the induced association of the FynT SH3 domain with SLAM-SAP was triggered by a change in the conformation of SLAM-associated SAP caused by SLAM engagement. Together, these data elucidate further the events initiating SLAM-SAP signaling in immune cells. Moreover, they identify a strictly inducible interaction mediated by an SH3 domain.
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Lavens, Delphine, Peter Ulrichts, Dominiek Catteeuw, Kris Gevaert, Joël Vandekerckhove, Frank Peelman, Sven Eyckerman, and Jan Tavernier. "The C-terminus of CIS defines its interaction pattern." Biochemical Journal 401, no. 1 (December 11, 2006): 257–67. http://dx.doi.org/10.1042/bj20060242.
Full textAbstract:
Proteins of the SOCS (suppressors of cytokine signalling) family are characterized by a conserved modular structure with pre-SH2 (Src homology 2), SH2 and SOCS-box domains. Several members, including CIS (cytokine-inducible SH2 protein), SOCS1 and SOCS3, are induced rapidly upon cytokine receptor activation and function in a negative-feedback loop, attenuating signalling at the receptor level. We used a recently developed mammalian two-hybrid system [MAPPIT (mammalian protein–protein interaction trap)] to analyse SOCS protein-interaction patterns in intact cells, allowing direct comparison with biological function. We find that, besides the SH2 domain, the C-terminal part of the CIS SOCS-box is required for functional interaction with the cytokine receptor motifs examined, but not with the N-terminal death domain of the TLR (Toll-like receptor) adaptor MyD88. Mutagenesis revealed that one single tyrosine residue at position 253 is a critical binding determinant. In contrast, substrate binding by the highly related SOCS2 protein, and also by SOCS1 and SOCS3, does not require their SOCS-box.
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Isakov, N., R. L. Wange, W. H. Burgess, J. D. Watts, R. Aebersold, and L. E. Samelson. "ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity." Journal of Experimental Medicine 181, no. 1 (January 1, 1995): 375–80. http://dx.doi.org/10.1084/jem.181.1.375.
Full textAbstract:
Engagement of the T cell antigen receptor (TCR) results in activation of several tyrosine kinases leading to tyrosine phosphorylation of protein substrates and activation of multiple biochemical pathways. TCR-mediated activation of the src-family kinases, Lck and Fyn, results in tyrosine phosphorylation of the TCR zeta and CD3 chains. The site of phosphorylation in these chains is the tyrosine-based activation motif (TAM), a 15-16 amino acid module containing two tyrosine residues. Tyrosine-phosphorylated TAMs serve as targets for binding of the zeta-associated protein (ZAP-70) tyrosine kinase via its tandem SH2 domains. This binding correlates with activation of ZAP-70, a critical event in T cell activation. To further define the structural requirements for ZAP-70 interaction with the TCR, we developed a binding assay using immobilized glutathione S-transferase fusion proteins containing the NH2- and/or COOH-terminal SH2 domains of ZAP-70, and soluble synthetic peptides with the sequence of the cytoplasmic region of the TCR zeta chain (TCR zeta cyt) or individual TCR zeta and CD3 epsilon TAM motifs. Direct binding studies demonstrated that the tandem ZAP-70 SH2 domains bind phosphorylated, but not nonphosphorylated, TCR zeta cyt. The NH2-terminal ZAP-70 SH2 domain also binds to TCR zeta cyt but with 100-fold lower affinity. No binding was observed with the COOH-terminal ZAP-70 SH2 domain. Similar studies demonstrated that the ZAP-70 tandem SH2 domain can bind a TCR zeta 3 TAM peptide in which both tyrosine residues are phosphorylated: Little or no binding was observed with peptides phosphorylated at only one tyrosine residue, or a nonphosphorylated peptide. Binding of the tandem SH2 domains to the other two TCR zeta TAM peptides and to a CD3 epsilon TAM peptide was also observed. All four doubly tyrosine phosphorylated TAM peptides cross-compete with each other for binding to the tandem SH2 domains of ZAP-70. The affinity of these peptides for the tandem SH2 construct demonstrated a hierarchy of TAM zeta 1 > or = TAM zeta 2 > TAM epsilon > or = TAM zeta 3. The results provide further evidence that the ZAP-70 interaction with the TCR requires prior phosphorylation of both tyrosine residues within a TAM motif. Binding of ZAP-70 to phospho-TAMs is notable for the high level of cooperativity between the two SH2 domains, which individually demonstrate low affinity interaction with the ligand. The cooperativity ensures higher affinity for the doubly phosphorylated ligand. Affinity differences of as much as 30-fold indicates a significant specificity of interaction of ZAP-70 SH2 domains for different phospho-TAMs.
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Meng, Li, JinPing Luo, Chunhua Li, and William H. Kinsey. "Role of Src homology 2 domain-mediated PTK signaling in mouse zygotic development." Reproduction 132, no. 3 (September 2006): 413–21. http://dx.doi.org/10.1530/rep.1.01151.
Full textAbstract:
Fyn and other Src-family kinases play an essential role at several steps during egg activation following fertilization of externally fertilizing species, such as marine invertebrates, fish, and frogs. Recent studies demonstrate that the requirement for Src-family kinases in activation of the mammalian egg is different from lower species, and the objective of this study was to test the role of the Fyn kinase in the mouse egg activated by intracytoplasmic sperm injection (ICSI). An Src homology 2 (SH2) domain containing fusion protein was used to suppress Fyn function in the mouse zygote following ICSI. Eggs injected with the Fyn SH2 domain at an intracellular concentration of 4–8 μM exhibited reduced developmental potential with 100% of the zygotes being arrested following the first or the second cleavage. At higher concentrations, the protein blocked pronuclear congression and the zygotes remained at the pronuclear stage. The SH2 domain had no effect on sperm-induced calcium oscillations in distinct contrast to its effect on the eggs of lower species. The results indicate that the SH2 domain of Fyn kinase plays an important role in pronuclear congression as well as early cleavage events and that this effect appears not to involve disruption of calcium oscillations.
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Nika, Konstantina, Lutz Tautz, Yutaka Arimura, Torkel Vang, Scott Williams, and Tomas Mustelin. "A Weak Lck Tail Bite Is Necessary for Lck Function in T Cell Antigen Receptor Signaling." Journal of Biological Chemistry 282, no. 49 (September 26, 2007): 36000–36009. http://dx.doi.org/10.1074/jbc.m702779200.
Full textAbstract:
Src family kinases are suppressed by a “tail bite” mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH) 2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)505 affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.
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Howell, B. W., and J. A. Cooper. "Csk suppression of Src involves movement of Csk to sites of Src activity." Molecular and Cellular Biology 14, no. 8 (August 1994): 5402–11. http://dx.doi.org/10.1128/mcb.14.8.5402-5411.1994.
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Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.
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Howell, B. W., and J. A. Cooper. "Csk suppression of Src involves movement of Csk to sites of Src activity." Molecular and Cellular Biology 14, no. 8 (August 1994): 5402–11. http://dx.doi.org/10.1128/mcb.14.8.5402.
Full textAbstract:
Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.
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Tanaka, M., R. Gupta, and B. J. Mayer. "Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins." Molecular and Cellular Biology 15, no. 12 (December 1995): 6829–37. http://dx.doi.org/10.1128/mcb.15.12.6829.
Full textAbstract:
SH2/SH3 adapters are thought to function in signal transduction pathways by coupling inputs from tyrosine kinases to downstream effectors such as Ras. Members of the mitogen-activated protein kinase family are known to be activated by a variety of mitogenic stimuli, including tyrosine kinases such as Abl and the epidermal growth factor (EGF) receptor. We have used activation of the mitogen-activated protein kinase Erk-1 as a model system with which to examine whether various dominant-negative SH2/SH3 adapters (Grb2, Crk, and Nck) could block signaling pathways leading to Erk activation. Activation of Erk-1 by oncogenic Abl was effectively inhibited by Grb2 with mutations in either its SH2 or SH3 domain or by Crk-1 with an SH3 domain mutation. The Crk-1 SH2 mutant was less effective, while Nck SH2 and SH3 mutants had little or no effect on Erk activation. These results suggest that both Crk and Grb2 may contribute to the activation of Erk by oncogenic Abl, whereas Nck is unlikely to participate in this pathway. Next we examined whether combinations of these dominant-negative adapters could inhibit Erk activation more effectively than each mutant alone. When combinations of Crk-1 and Grb2 mutants were analyzed, the combination of the Crk-1 SH3 mutant plus the Grb2 SH3 mutant gave a striking synergistic effect. This finding suggests that in Abl-transformed cells, more than one class of tyrosine-phosphorylated sites (those that bind the Grb2 SH2 domain and those that bind the Crk SH2 domain) can lead to Ras activation. In contrast to results with Abl, Erk activation by EGF was strongly inhibited only by Grb2 mutants; Crk and Nck mutants had little or no effect. This finding suggests that Grb2 is the only adapter involved in the activation of Erk by EGF. Dominant-negative adaptors provide a novel means to identify binding interactions important in vivo for signaling in response to a variety of stimuli.
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HELLYER, Nathan J., Kunrong CHENG, and John G. KOLAND. "ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase." Biochemical Journal 333, no. 3 (August 1, 1998): 757–63. http://dx.doi.org/10.1042/bj3330757.
Full textAbstract:
ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr → Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.
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Yang, Edward, Zilong Wen, Richard L. Haspel, Jue J. Zhang, and James E. Darnell. "The Linker Domain of Stat1 Is Required for Gamma Interferon-Driven Transcription." Molecular and Cellular Biology 19, no. 7 (July 1, 1999): 5106–12. http://dx.doi.org/10.1128/mcb.19.7.5106.
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ABSTRACT Upon binding of gamma interferon (IFN-γ) to its receptor, the latent transcription factor Stat1 becomes phosphorylated, dimerizes, and enters the nucleus to activate transcription. In response to IFN-α, Stat1 binds to Stat2 in a heterodimer that recruits p48, an IRF family member, to activate transcription. A number of functional domains of the STATs, including a C-terminal transactivation domain, a dimerization domain, and an SH2 domain, are known. However, the highly conserved residues between the DNA binding and SH2 domains (463 to 566), recently christened the linker domain on the basis of crystallographic studies, have remained without a known function. In the present study, we report that KE544-545AA point mutants in Stat1 abolish transcriptional responses to IFN-γ but not to IFN-α. We further show that this mutant Stat1 undergoes normal phosphorylation, nuclear translocation, and DNA binding. Taken together with recent structural evidence, these results suggest that the linker domain acts as a critical contact point during the construction of a Stat1-driven transcriptional complex.
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Campbell, S. J., and R. M. Jackson. "Diversity in the SH2 domain family phosphotyrosyl peptide binding site." Protein Engineering, Design and Selection 16, no. 3 (March 2003): 217–27. http://dx.doi.org/10.1093/proeng/gzg025.
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Liao, Yi-Chun, Lizhen Si, Ralph W. deVere White, and Su Hao Lo. "The phosphotyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1." Journal of Cell Biology 176, no. 1 (December 26, 2006): 43–49. http://dx.doi.org/10.1083/jcb.200608015.
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The tensin family member cten (C-terminal tensin like) is an Src homology 2 (SH2) and phosphotyrosine binding domain–containing focal adhesion molecule that may function as a tumor suppressor. However, the mechanism has not been well established. We report that cten binds to another tumor suppressor, deleted in liver cancer 1 (DLC-1), and the SH2 domain of cten is responsible for the interaction. Unexpectedly, the interaction between DLC-1 and the cten SH2 domain is independent of tyrosine phosphorylation of DLC-1. By site-directed mutagenesis, we have identified several amino acid residues on cten and DLC-1 that are essential for this interaction. Mutations on DLC-1 perturb the interaction with cten and disrupt the focal adhesion localization of DLC-1. Furthermore, these DLC-1 mutants have lost their tumor suppression activities. When these DLC-1 mutants were fused to a focal adhesion targeting sequence, their tumor suppression activities were significantly restored. These results provide a novel mechanism whereby the SH2 domain of cten-mediated focal adhesion localization of DLC-1 plays an essential role in its tumor suppression activity.
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Jahn, Thomas, Petra Seipel, Susanne Urschel, Christian Peschel, and Justus Duyster. "Role for the Adaptor Protein Grb10 in the Activation of Akt." Molecular and Cellular Biology 22, no. 4 (February 15, 2002): 979–91. http://dx.doi.org/10.1128/mcb.22.4.979-991.2002.
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ABSTRACT Grb10 is a member of the Grb7 family of adapter proteins lacking intrinsic enzymatic function and encodes functional domains including a pleckstrin homology (PH) domain and an SH2 domain. The role of different Grb10 splice variants in signal transduction of growth factors like insulin or insulin-like growth factor has been described as inhibitory or stimulatory depending on the presence of a functional PH and/or SH2 domain. Performing a yeast two-hybrid screen with the c-kit cytoplasmic tail fused to LexA as a bait and a mouse embryo cDNA library as prey, we found that the Grb10 SH2 domain interacted with the c-kit receptor tyrosine kinase. In the course of SCF-mediated activation of c-kit, Grb10 is recruited to the c-kit receptor in an SH2 domain- and phosphotyrosine-dependent but PH domain-independent manner. We found that Akt and Grb10 form a constitutive complex, suggesting a role for Grb10 in the translocation of Akt to the cell membrane. Indeed, coexpression studies revealed that Grb10 and c-kit activate Akt in a synergistic manner. This dose-dependent effect of Grb10 is wortmannin sensitive and was also seen at a lower level in cells in which c-kit was not expressed. Expression of a Grb10 mutant lacking the SH2 domain as well as a mutant lacking the PH domain did not influence Akt activity. Grb10-induced Akt activation was observed without increased phosphatidylinositol 3-kinase (PI3-kinase) activity, suggesting that Grb10 is a positive regulator of Akt downstream of PI3-kinase. Significantly, deficient activation of Akt by a constitutively activated c-kit mutant lacking the binding site for PI3-kinase (c-kitD814V/Y719F) could be fully compensated by overexpression of Grb10. In Ba/F3 cells, the incapacity of c-kitD814V/Y719F to induce interleukin-3 (IL-3)-independent growth could be rescued by overexpression of Grb10. In contrast, expression of the SH2 deletion mutant of Grb10 together with c-kitD814V/Y719F did not render Ba/F3 cells independent of IL-3. In summary, we provide evidence that Grb10 is part of the c-kit signaling pathway and that the expression level of Grb10 critically influences Akt activity. We propose a model in which Grb10 acts as a coactivator for Akt by virtue of its ability to form a complex with Akt and its SH2 domain-dependent translocation to the cell membrane.
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Zhang, Tong, Wei Hua Kee, Kah Tong Seow, Winnie Fung, and Xinmin Cao. "The Coiled-Coil Domain of Stat3 Is Essential for Its SH2 Domain-Mediated Receptor Binding and Subsequent Activation Induced by Epidermal Growth Factor and Interleukin-6." Molecular and Cellular Biology 20, no. 19 (October 1, 2000): 7132–39. http://dx.doi.org/10.1128/mcb.20.19.7132-7139.2000.
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ABSTRACT STAT proteins are a family of latent transcription factors that mediate the response to various cytokines and growth factors. Upon stimulation by cytokines, STAT proteins are recruited to the receptors via their SH2 domains, phosphorylated on a specific tyrosine, dimerized, and translocated into the nucleus, where they bind specific DNA sequences and activate the target gene transcription. STATs share highly conserved structures, including an N-domain, a coiled-coil domain, a DNA-binding domain, a linker domain, and an SH2 domain. To investigate the role of the coiled-coil domain, we performed a systematic deletion analysis of the N-domain and each of the α-helices and mutagenesis of conserved residues in the coiled-coil region of Stat3. Our results indicate that the coiled-coil domain is essential for Stat3 recruitment to the receptor and the subsequent tyrosine phosphorylation and tyrosine phosphorylation-dependent activities, such as dimer formation, nuclear translocation, and DNA binding, stimulated by epidermal growth factor (EGF) or interleukin-6 (IL-6). Single mutation of Asp170 or, to a lesser extent, Lys177 in α-helix 1 diminishes both receptor binding and tyrosine phosphorylation. Furthermore, the Asp170 mutant retains its ability to bind to DNA when phosphorylated on Tyr705 by Src kinase in vitro, implying a functional SH2 domain. Finally, we demonstrate a direct binding of Stat3 to the receptor. Taken together, our data reveal a novel role for the coiled-coil domain that regulates the early events in Stat3 activation and function.
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Courtneidge, Sara A., Stefano Fumagalli, Manfred Koegl, Giulio Superti-Furga, and Geraldine M. Twamley-Stein. "The Src family of protein tyrosine kinases: regulation and functions." Development 119, Supplement (December 1, 1993): 57–64. http://dx.doi.org/10.1242/dev.119.supplement.57.
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Most of the nine members of the Src family of tyrosine kinases arc restricted in their expression, often to cells of the haematopoietic lineage, while some, particularly Src, Fyn and Yes, are more unbiquitously expressed. We have been studying the functions of Src, Fyn and Yes in fibroblasts. We have shown that stimulation of quiescent fibroblasts with platelet-derived growth factor (PDGF) causes Src, Fyn and Yes to become activated, and to associate transiently with the PDGF receptor. To address the role of Src, Fyn and Yes in the response to PDGF, we have used a dominant negative approach, in which cells were engineered to express catalytically inactive forms of Src kinases. These cells were unable to enter S phase in response to PDGF, and we therefore conclude that Src family tyrosine kinases are required in order for the PDGF receptor to transmit a mitogenic signal. It has previously been shown that the kinase activity of Src is negatively regulated by phosphorylation of tyr 527 in its carboxy-terminal tail. A kinase, Csk, that phosphorylates tyr 527 has recently been identified. We expressed Src in yeast to test the model that phosphorylation of tyr 527 represses activity by promoting intramolecular association between the tail and the SH2 domain. Inducible expression of Src in S. pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in the SH2 domain were as lethal as wild-type Src, but were insensitive to Csk. We interpret these results in favour of an SH2 domain : phosphorylated tail interaction repressing Src activity. However, we have also found that Src molecules containing mutations in the SH3 domain are not regulated by Csk, suggesting that the SH3 domain also functions in the intramolecular regulation of Src activity.
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Wolf, Ingrid, Brendan J. Jenkins, Yan Liu, Martina Seiffert, Joseph M. Custodio, Paul Young, and Larry R. Rohrschneider. "Gab3, a New DOS/Gab Family Member, Facilitates Macrophage Differentiation." Molecular and Cellular Biology 22, no. 1 (January 1, 2002): 231–44. http://dx.doi.org/10.1128/mcb.22.1.231-244.2002.
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ABSTRACT Using the FDC-P1 cell line expressing the exogenous macrophage colony-stimulating factor (M-CSF) receptor, Fms, we have analyzed the role of a new mammalian DOS/Gab-related signaling protein, called Gab3, in macrophage cell development of the mouse. Gab3 contains an amino-terminal pleckstrin homology domain, multiple potential sites for tyrosine phosphorylation and SH2 domain binding, and two major polyproline motifs potentially interacting with SH3 domains. Among the growing family of Gab proteins, Gab3 exhibits a unique and overlapping pattern of expression in tissues of the mouse compared with Gab1 and Gab2. Gab3 is more restricted to the hematopoietic tissues such as spleen and thymus but is detectable at progressively lower levels within heart, kidney, uterus, and brain. Like Gab2, Gab3 is tyrosine phosphorylated after M-CSF receptor stimulation and associates transiently with the SH2 domain-containing proteins p85 and SHP2. Overexpression of exogenous Gab3 in FD-Fms cells dramatically accelerates macrophage differentiation upon M-CSF stimulation. Unlike Gab2, which shows a constant mRNA expression level after M-CSF stimulation, Gab3 expression is initially absent or low in abundance in FD cells expressing the wild-type Fms, but Gab3 mRNA levels are increased upon M-CSF stimulation. Moreover, M-CSF stimulation of FD-FmsY807F cells (which grow but do not differentiate) fails to increase Gab3 expression. These results suggest that Gab3 is important for macrophage differentiation and that differentiation requires the early phosphorylation of Gab2 followed by induction and subsequent phosphorylation of Gab3.