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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.
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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.
2
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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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.
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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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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.
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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.
5
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.
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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.
6
Tian, M., and G. S. Martin. "The role of the Src homology domains in morphological transformation by v-src." Molecular Biology of the Cell 8, no. 7 (July 1997): 1183–93. http://dx.doi.org/10.1091/mbc.8.7.1183.
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The Src homology (SH2 and SH3) domains of v-Src are required for transformation of Rat-2 cells and for wild-type (morphr) transformation of chicken embryo fibroblasts (CEFs). We report herein that the N-terminal domains of v-Src, when expressed in trans, cannot complement the transformation defect of a deletion mutant lacking the "unique," SH3, and SH2 regions. However, the same regions of Src can promote transformation when translocated to the C terminus of v-Src, although the transformation of CEFs is somewhat slower. We conclude that the SH3 and SH2 domains must be present in cis to the catalytic domain to promote transformation but that transformation is not dependent on the precise intramolecular location of these domains. In CEFSs and in Rat-2 cells, the expression of wild-type v-Src results in tyrosine phosphorylation of proteins that bind to the v-Src SH3 and SH2 domains in vitro; mutations in the SH2 or SH3 and SH2 domains prevent the phosphorylation of these proteins. These findings are most consistent with models in which the SH3 and SH2 domains of v-Src directly or indirectly target the catalytic domain to substrates involved in transformation. However, the N-terminal domains of v-Src can promote tyrosine phosphorylation of certain proteins, in particular p130Cas, even when expressed in the absence of the catalytic domain, indicating that the N-terminal domains of v-Src have effects that are independent of the catalytic domain.
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DeBell, Karen E., Bogdan A. Stoica, Maria-Concetta Verí, Angela Di Baldassarre, Sebastiano Miscia, Laurie J. Graham, Barbara L. Rellahan, Masamichi Ishiai, Tomohiro Kurosaki, and Ezio Bonvini. "Functional Independence and Interdependence of the Src Homology Domains of Phospholipase C-γ1 in B-Cell Receptor Signal Transduction." Molecular and Cellular Biology 19, no. 11 (November 1, 1999): 7388–98. http://dx.doi.org/10.1128/mcb.19.11.7388.
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ABSTRACT B-cell receptor (BCR)-induced activation of phospholipase C-γ1 (PLCγ1) and PLCγ2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCγ activation, the mechanism coupling PLCγ to the BCR remains undefined. The role of PLCγ1 SH2 and SH3 domains at different steps of BCR-induced PLCγ1 activation was examined by reconstitution in a PLCγ-negative B-cell line. PLCγ1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCγ1 with the adapter protein, BLNK. Forcing PLCγ1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCγ1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCγ1 activation.
8
Sipeki, Szabolcs, Kitti Koprivanacz, Tamás Takács, Anita Kurilla, Loretta László, Virag Vas, and László Buday. "Novel Roles of SH2 and SH3 Domains in Lipid Binding." Cells 10, no. 5 (May 13, 2021): 1191. http://dx.doi.org/10.3390/cells10051191.
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Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.
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Tu, Yizeng, Fugang Li, and Chuanyue Wu. "Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways." Molecular Biology of the Cell 9, no. 12 (December 1998): 3367–82. http://dx.doi.org/10.1091/mbc.9.12.3367.
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Many of the protein–protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain–containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways.
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Kazemein Jasemi, Neda S., Christian Herrmann, Eva Magdalena Estirado, Lothar Gremer, Dieter Willbold, Luc Brunsveld, Radovan Dvorsky, and Mohammad R. Ahmadian. "The intramolecular allostery of GRB2 governing its interaction with SOS1 is modulated by phosphotyrosine ligands." Biochemical Journal 478, no. 14 (July 23, 2021): 2793–809. http://dx.doi.org/10.1042/bcj20210105.
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Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g. HER2). The elucidation of structural organization and mechanistic insights into GRB2 interactions, however, remain challenging due to their inherent flexibility. This study represents an important advance in our mechanistic understanding of how GRB2 links RTKs to SOS1. Accordingly, it can be proposed that (1) HER2 pYP-bound SH2 potentiates GRB2 SH3 domain interactions with SOS1 (an allosteric mechanism); (2) the SH2 domain blocks cSH3, enabling nSH3 to bind SOS1 first before cSH3 follows (an avidity-based mechanism); and (3) the allosteric behavior of cSH3 to other domains appears to be unidirectional, although there is an allosteric effect between the SH2 and SH3 domains.
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Smith, Jesse J., D. Ashley Richardson, Jan Kopf, Minoru Yoshida, Robert E. Hollingsworth, and Sally Kornbluth. "Apoptotic Regulation by the Crk Adapter Protein Mediated by Interactions with Wee1 and Crm1/Exportin." Molecular and Cellular Biology 22, no. 5 (March 1, 2002): 1412–23. http://dx.doi.org/10.1128/mcb.22.5.1412-1423.2002.
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ABSTRACT The adapter protein Crk contains an SH2 domain and two SH3 domains. Through binding of particular ligands to the SH2 domain and the N-terminal SH3 domain, Crk has been implicated in a number of signaling processes, including regulation of cell growth, cell motility, and apoptosis. We report here that the C-terminal SH3 domain, never shown to bind any specific signaling molecules, contains a binding site for the nuclear export factor Crm1. We find that a mutant Crk protein, deficient in Crm1 binding, promotes apoptosis. Moreover, this nuclear export sequence mutant [NES(−) Crk] interacts strongly, through its SH2 domain, with the nuclear tyrosine kinase, Wee1. Collectively, these data suggest that a nuclear population of Crk bound to Wee1 promotes apoptotic death of mammalian cells.
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Medema, R. H., W. L. de Laat, G. A. Martin, F. McCormick, and J. L. Bos. "GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion." Molecular and Cellular Biology 12, no. 8 (August 1992): 3425–30. http://dx.doi.org/10.1128/mcb.12.8.3425.
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The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.
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Medema, R. H., W. L. de Laat, G. A. Martin, F. McCormick, and J. L. Bos. "GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion." Molecular and Cellular Biology 12, no. 8 (August 1992): 3425–30. http://dx.doi.org/10.1128/mcb.12.8.3425-3430.1992.
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The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.
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Pawson, Tony. "SH2 and SH3 domains." Current Opinion in Structural Biology 2, no. 3 (June 1992): 432–37. http://dx.doi.org/10.1016/0959-440x(92)90235-y.
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Pawson, Tony. "SH2 and SH3 domains." Current Biology 2, no. 6 (June 1992): 299–300. http://dx.doi.org/10.1016/0960-9822(92)90873-9.
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Pawson, T., and J. Schlessingert. "SH2 and SH3 domains." Current Biology 3, no. 7 (July 1993): 434–42. http://dx.doi.org/10.1016/0960-9822(93)90350-w.
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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.
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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.14.8.5402-5411.1994.
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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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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.
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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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Prasad, K. V., R. Kapeller, O. Janssen, H. Repke, J. S. Duke-Cohan, L. C. Cantley, and C. E. Rudd. "Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase." Molecular and Cellular Biology 13, no. 12 (December 1993): 7708–17. http://dx.doi.org/10.1128/mcb.13.12.7708.
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CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.
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Prasad, K. V., R. Kapeller, O. Janssen, H. Repke, J. S. Duke-Cohan, L. C. Cantley, and C. E. Rudd. "Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase." Molecular and Cellular Biology 13, no. 12 (December 1993): 7708–17. http://dx.doi.org/10.1128/mcb.13.12.7708-7717.1993.
Full textAbstract:
CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.
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Flynn, D. C., T. H. Leu, A. B. Reynolds, and J. T. Parsons. "Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate." Molecular and Cellular Biology 13, no. 12 (December 1993): 7892–900. http://dx.doi.org/10.1128/mcb.13.12.7892.
Full textAbstract:
Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.
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Flynn, D. C., T. H. Leu, A. B. Reynolds, and J. T. Parsons. "Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate." Molecular and Cellular Biology 13, no. 12 (December 1993): 7892–900. http://dx.doi.org/10.1128/mcb.13.12.7892-7900.1993.
Full textAbstract:
Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.
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Cooper, J. A., and A. Kashishian. "In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase." Molecular and Cellular Biology 13, no. 3 (March 1993): 1737–45. http://dx.doi.org/10.1128/mcb.13.3.1737.
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We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cooper, J. A., and A. Kashishian. "In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase." Molecular and Cellular Biology 13, no. 3 (March 1993): 1737–45. http://dx.doi.org/10.1128/mcb.13.3.1737-1745.1993.
Full textAbstract:
We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
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Russell, Robert B., and Geoffrey J. Barton. "An SH2—SH3 domain hybrid." Nature 364, no. 6440 (August 1993): 765. http://dx.doi.org/10.1038/364765a0.
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RIORDAN, Stephen M., Sukhwinderjit LIDDER, Roger WILLIAMS, and George G. SKOUTERIS. "The β-subunit of the hepatocyte growth factor/scatter factor (HGF/SF) receptor phosphorylates and associates with CrkII: expression of CrkII enhances HGF/SF-induced mitogenesis." Biochemical Journal 350, no. 3 (September 8, 2000): 925–32. http://dx.doi.org/10.1042/bj3500925.
Full textAbstract:
CrkII, a 40kDa adaptor possessing a Src homology (SH)2 domain followed by two SH3 domains, although not endowed with catalytic activity, participates in intracellular signalling, presumably by activating the Ras pathway. CrkII was found to be phosphorylated in response to hepatocyte growth factor/scatter factor (HGF/SF) and to associate with the beta-subunit of the HGF receptor (MET). CrkII associated with p145βMET via its SH2 domain. Growth-factor-receptor-bound protein 2 (Grb2) co-immunoprecipitated with CrkII species. By transient transfection of A431 human epidermoid carcinoma cells with wild-type and dominant-negative Grb2 expression constructs lacking either the SH2 or SH3 domains, we have concluded that Grb2 does not contribute to the ‘presentation’ of CrkII to p145βMET. Overexpression of wild-type CrkII in A431 cells enhanced HGF/SF-induced proliferation, while a CrkII dominant-negative mutant lacking the SH2 domain prevented a similar proliferating response to HGF/SF. The effect of CrkII on HGF/SF-induced proliferation was also abolished in cells co-expressing CrkII and Son-of-sevenless lacking the guanine exchange domain, suggesting that CrkII is likely to induce cell proliferation partly via the Ras/mitogen-activated protein kinase route.
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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.
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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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She, H. Y., S. Rockow, J. Tang, R. Nishimura, E. Y. Skolnik, M. Chen, B. Margolis, and W. Li. "Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells." Molecular Biology of the Cell 8, no. 9 (September 1997): 1709–21. http://dx.doi.org/10.1091/mbc.8.9.1709.
Full textAbstract:
Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton.
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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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Liu, X., L. E. Marengere, C. A. Koch, and T. Pawson. "The v-Src SH3 domain binds phosphatidylinositol 3'-kinase." Molecular and Cellular Biology 13, no. 9 (September 1993): 5225–32. http://dx.doi.org/10.1128/mcb.13.9.5225.
Full textAbstract:
Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.
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Liu, X., L. E. Marengere, C. A. Koch, and T. Pawson. "The v-Src SH3 domain binds phosphatidylinositol 3'-kinase." Molecular and Cellular Biology 13, no. 9 (September 1993): 5225–32. http://dx.doi.org/10.1128/mcb.13.9.5225-5232.1993.
Full textAbstract:
Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.
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Tanaka, S., S. Hattori, T. Kurata, K. Nagashima, Y. Fukui, S. Nakamura, and M. Matsuda. "Both the SH2 and SH3 domains of human CRK protein are required for neuronal differentiation of PC12 cells." Molecular and Cellular Biology 13, no. 7 (July 1993): 4409–15. http://dx.doi.org/10.1128/mcb.13.7.4409.
Full textAbstract:
Human CRK protein is a homolog of the chicken v-crk oncogene product and consists mostly of src homology region 2 (SH2) and SH3, which are shared by many proteins, in particular those involved in signal transduction. SH2 has been shown to bind specifically to phosphotyrosine-containing peptides. We report here that both SH2 and SH3 are required for signaling from CRK protein. Microinjection of the CRK protein induced neurite formation of rat pheochromocytoma cell line PC12. This activity was abolished by mutation of the CRK protein in either SH2 or SH3. The neuronal differentiation induced by the CRK protein was blocked by an excess amount of peptides containing CRK SH3. Moreover, we identified three proteins, of 118, 125, and 136 kDa, which bound specifically to CRK SH3. The CRK-induced neuronal differentiation was also suppressed by monoclonal antibodies against either CRK SH2 or p21ras. These results suggest that both SH2 and SH3 of the CRK protein mediate specific protein-protein binding and that the resulting multimolecular complex generates a signal for neurite differentiation through activation of p21ras.
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Tanaka, S., S. Hattori, T. Kurata, K. Nagashima, Y. Fukui, S. Nakamura, and M. Matsuda. "Both the SH2 and SH3 domains of human CRK protein are required for neuronal differentiation of PC12 cells." Molecular and Cellular Biology 13, no. 7 (July 1993): 4409–15. http://dx.doi.org/10.1128/mcb.13.7.4409-4415.1993.
Full textAbstract:
Human CRK protein is a homolog of the chicken v-crk oncogene product and consists mostly of src homology region 2 (SH2) and SH3, which are shared by many proteins, in particular those involved in signal transduction. SH2 has been shown to bind specifically to phosphotyrosine-containing peptides. We report here that both SH2 and SH3 are required for signaling from CRK protein. Microinjection of the CRK protein induced neurite formation of rat pheochromocytoma cell line PC12. This activity was abolished by mutation of the CRK protein in either SH2 or SH3. The neuronal differentiation induced by the CRK protein was blocked by an excess amount of peptides containing CRK SH3. Moreover, we identified three proteins, of 118, 125, and 136 kDa, which bound specifically to CRK SH3. The CRK-induced neuronal differentiation was also suppressed by monoclonal antibodies against either CRK SH2 or p21ras. These results suggest that both SH2 and SH3 of the CRK protein mediate specific protein-protein binding and that the resulting multimolecular complex generates a signal for neurite differentiation through activation of p21ras.
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Klippel, A., J. A. Escobedo, W. J. Fantl, and L. T. Williams. "The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor." Molecular and Cellular Biology 12, no. 4 (April 1992): 1451–59. http://dx.doi.org/10.1128/mcb.12.4.1451.
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Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor.
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Klippel, A., J. A. Escobedo, W. J. Fantl, and L. T. Williams. "The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor." Molecular and Cellular Biology 12, no. 4 (April 1992): 1451–59. http://dx.doi.org/10.1128/mcb.12.4.1451-1459.1992.
Full textAbstract:
Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor.
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Shvartsman, Dmitry E., John C. Donaldson, Begoña Diaz, Orit Gutman, G. Steven Martin, and Yoav I. Henis. "Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets." Journal of Cell Biology 178, no. 4 (August 13, 2007): 675–86. http://dx.doi.org/10.1083/jcb.200701133.
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Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src–GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src.
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Moore, B., G. Seroussi, and B. Jochim. "Explorer de nouveaux domaines SH2/SH3." médecine/sciences 9, no. 6-7 (1993): 788. http://dx.doi.org/10.4267/10608/2993.
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Vihinen, Mauno, and C. I. Edvard Smith. "Interactions between SH2 and SH3 Domains." Biochemical and Biophysical Research Communications 242, no. 2 (January 1998): 351–56. http://dx.doi.org/10.1006/bbrc.1997.7909.
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Kuriyan, John, and David Cowburn. "Structures of SH2 and SH3 domains." Current Opinion in Structural Biology 3, no. 6 (January 1993): 828–37. http://dx.doi.org/10.1016/0959-440x(93)90145-b.
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Mayer, Bruce J., and David Baltimore. "Signalling through SH2 and SH3 domains." Trends in Cell Biology 3, no. 1 (January 1993): 8–13. http://dx.doi.org/10.1016/0962-8924(93)90194-6.
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Le, Ngocdiep, and Michael A. Simon. "Disabled Is a Putative Adaptor Protein That Functions during Signaling by the Sevenless Receptor Tyrosine Kinase." Molecular and Cellular Biology 18, no. 8 (August 1, 1998): 4844–54. http://dx.doi.org/10.1128/mcb.18.8.4844.
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ABSTRACT DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by thesevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.
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Okuda, K., A. D’Andrea, R. A. Van Etten, and J. D. Griffin. "The C-Terminus of c-Abl Is Required for Proliferation and Viability Signaling in a c-Abl/Erythropoietin Receptor Fusion Protein." Blood 92, no. 10 (November 15, 1998): 3848–56. http://dx.doi.org/10.1182/blood.v92.10.3848.
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Abstract Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (▵SH3), the SH2 domain was deleted (▵SH2), the C-terminal actin-binding domain was deleted (▵ABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the ▵SH3 mutant was equivalent, the ▵SH2 mutant was moderately impaired, and the ▵ABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and ▵SH3. However, time to death was prolonged by at least twofold for ▵SH2 and greater than threefold for ▵ABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.
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Okuda, K., A. D’Andrea, R. A. Van Etten, and J. D. Griffin. "The C-Terminus of c-Abl Is Required for Proliferation and Viability Signaling in a c-Abl/Erythropoietin Receptor Fusion Protein." Blood 92, no. 10 (November 15, 1998): 3848–56. http://dx.doi.org/10.1182/blood.v92.10.3848.422k44_3848_3856.
Full textAbstract:
Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (▵SH3), the SH2 domain was deleted (▵SH2), the C-terminal actin-binding domain was deleted (▵ABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the ▵SH3 mutant was equivalent, the ▵SH2 mutant was moderately impaired, and the ▵ABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and ▵SH3. However, time to death was prolonged by at least twofold for ▵SH2 and greater than threefold for ▵ABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.
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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.
Full textAbstract:
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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Matsuda, M., B. J. Mayer, and H. Hanafusa. "Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins." Molecular and Cellular Biology 11, no. 3 (March 1991): 1607–13. http://dx.doi.org/10.1128/mcb.11.3.1607.
Full textAbstract:
The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.
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Matsuda, M., B. J. Mayer, and H. Hanafusa. "Identification of domains of the v-crk oncogene product sufficient for association with phosphotyrosine-containing proteins." Molecular and Cellular Biology 11, no. 3 (March 1991): 1607–13. http://dx.doi.org/10.1128/mcb.11.3.1607-1613.1991.
Full textAbstract:
The oncogene product of the avian sarcoma virus CT10, P47gag-crk, contains the SH2, SH2', and SH3 domains and binds proteins in a phosphotyrosine (ptyr)-dependent manner. In this study, we have determined the region of P47gag-crk essential for binding to ptyr-containing proteins. Mutant P47gag-crk proteins expressed in Escherichia coli that have the intact SH2 and SH2' regions retained the capacity to bind ptyr-containing proteins obtained from cells transformed by crk and src. The deletion of SH2 resulted in the loss of binding activity. Other mutants that have altered SH2 or SH2' bound few, if any, of the ptyr-containing proteins. Those mutants that bound ptyr-containing proteins associated with tyrosine kinase activity. We also found that polypeptides containing SH2, SH2', and SH3 of p60v-src and p60c-src associated with ptyr-containing proteins from crk-transformed cells. Thus, the SH2 and SH2' domains of P47gag-crk are responsible for their binding to ptyr-containing proteins.
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Xing, Z., H. C. Chen, J. K. Nowlen, S. J. Taylor, D. Shalloway, and J. L. Guan. "Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain." Molecular Biology of the Cell 5, no. 4 (April 1994): 413–21. http://dx.doi.org/10.1091/mbc.5.4.413.
Full textAbstract:
The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.
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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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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.