Journal articles on the topic 'RasGAP SH3 domain binding proteins'
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1
Cong, Feng, Bing Yuan, and Stephen P. Goff. "Characterization of a Novel Member of the DOK Family That Binds and Modulates Abl Signaling." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 8314–25. http://dx.doi.org/10.1128/mcb.19.12.8314.
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ABSTRACT A novel member of the p62 dok family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62 dok bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62 dok , DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62 dok , suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function.
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Klinghoffer, R. A., B. Duckworth, M. Valius, L. Cantley, and A. Kazlauskas. "Platelet-derived growth factor-dependent activation of phosphatidylinositol 3-kinase is regulated by receptor binding of SH2-domain-containing proteins which influence Ras activity." Molecular and Cellular Biology 16, no. 10 (October 1996): 5905–14. http://dx.doi.org/10.1128/mcb.16.10.5905.
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Upon binding of platelet-derived growth factor (PDGF), the PDGF beta receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and binds several SH2-domain-containing signal relay enzymes, including phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase SHP-2. In this study, we have investigated whether PDGF-dependent PI3K activation is affected by the other proteins that associate with the PDGFR. We constructed and characterized a series of PDGFR mutants which contain binding sites for PI3K as well as one additional protein, either RasGAP, SHP-2, or PLC gamma. While all of the receptors had wild-type levels of PDGF-stimulated tyrosine kinase activity and associated with comparable amounts of PI3K activity, their abilities to trigger accumulation of PI3K products in vivo differed dramatically. The wild-type receptor, as well as receptors that recruited PI3K or PI3K and SHP-2, were all capable of fully activating PI3K. In contrast, receptors that associated with PI3K and RasGAP or PI3K and PLC gamma displayed a greatly reduced ability to stimulate production of PI3K products. When this series of receptors was tested for their ability to activate Ras, we observed a strong positive correlation between Ras activation and PI3K activation. Further investigation of the relationship between Ras and PI3K indicated that Ras was upstream of PI3K. Thus, activation of PI3K requires not only binding of PI3K to the tyrosine-phosphorylated PDGFR but accumulation of GTP-bound Ras as well. Furthermore, PLC gamma and RasGAP negatively modulate PDGF-dependent PI3K activation. Finally, PDGF-stimulated signal relay can be regulated by altering the ratio of SH2-domain-containing enzymes that are recruited to the PDGFR.
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Solomon, Samuel, Yaoxian Xu, Bin Wang, Muriel D. David, Peter Schubert, Derek Kennedy, and John W. Schrader. "Distinct Structural Features ofCaprin-1 Mediate Its Interaction with G3BP-1 and Its Induction of Phosphorylation of Eukaryotic Translation InitiationFactor 2α, Entry to Cytoplasmic Stress Granules, and Selective Interaction with a Subset of mRNAs." Molecular and Cellular Biology 27, no. 6 (January 8, 2007): 2324–42. http://dx.doi.org/10.1128/mcb.02300-06.
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ABSTRACT Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G1-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2α (eIF-2α) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.
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Gallouzi, Imed-eddine, Fabienne Parker, Karim Chebli, Florence Maurier, Emmanuel Labourier, Isabelle Barlat, Jean-Paul Capony, Bruno Tocque, and Jamal Tazi. "A Novel Phosphorylation-Dependent RNase Activity of GAP-SH3 Binding Protein: a Potential Link between Signal Transduction and RNA Stability." Molecular and Cellular Biology 18, no. 7 (July 1, 1998): 3956–65. http://dx.doi.org/10.1128/mcb.18.7.3956.
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ABSTRACT A potential p120 GTPase-activating protein (RasGAP) effector, G3BP (RasGAP Src homology 3 [SH3] binding protein), was previously identified based on its ability to bind the SH3 domain of RasGAP. Here we show that G3BP colocalizes and physically interacts with RasGAP at the plasma membrane of serum-stimulated but not quiescent Chinese hamster lung fibroblasts. In quiescent cells, G3BP was hyperphosphorylated on serine residues, and this modification was essential for its activity. Indeed, G3BP harbors a phosphorylation-dependent RNase activity which specifically cleaves the 3′-untranslated region of human c-myc mRNA. The endoribonuclease activity of G3BP can initiate mRNA degradation and therefore represents a link between a RasGAP-mediated signaling pathway and RNA turnover.
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Patsoukis, Nikolaos, Esther M. Lafuente, and Vassiliki A. Boussiotis. "RIAM and RapL Regulate Distinct Signaling Events and Functional Outcomes Upon TCR-Mediated Activation." Blood 114, no. 22 (November 20, 2009): 3683. http://dx.doi.org/10.1182/blood.v114.22.3683.3683.
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Abstract Abstract 3683 Poster Board III-619 Rap1, a small GTPase of the Ras superfamily, originally identified by its ability to reverse Ras-mediated transformation, is now known to regulate cytoskeletal reorganization, cell morphology, adherens junction positioning and adhesion. The best-studied function of Rap1 is inside-out activation of integrins and cell adhesion. Two newly identified Rap1 effectors, RapL and RIAM, have been implicated in Rap1 mediated in inside-out activation of integrins and cell adhesion. However, significant differences in the structure and interactions of these molecules indicate that they may mediate distinct signaling events. RIAM has a N-terminal coiled-coil region, central RA and pleckstrin homology (PH) domains, and proline-rich N-terminal and C-terminal regions, with multiple FPPPP motifs capable of interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP, multiple XPPPP motifs interacting with Profilin and multiple PXXP motifs capable of interacting with SH3 domain containing proteins. Because of these properties RIAM is a regulator of actin polymerization but also interacts with components of the T cell signaling machinery. In contrast, RapL has an RBD (Ras binding domain-structurally similar to RA domain) and a C-terminal coiled-coil region and interacts with Rap1 via its RBD domain and its N-terminal region, and with the aL subunit of LFA-1 via its C-terminal domain. In the present study we investigated the role of RIAM and RapL in regulating signaling and functional events activated via the TCR. For this purpose we used RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, had been depleted by siRNA. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by depletion of RIAM, activation of these kinases was unaffected by depletion of RapL. In contrast, activation of p38 was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. Moreover, RIAM knockdown resulted in impaired activation of Ras and Rap1 due to defective activation of the calcium and diacylglycerole-dependent GEFs, RasGRP1 and CalDAG-GEFI. In contrast, RapL knockdown had no effect on these events compared to control Jurkat T cells. Strikingly, RIAM-KD cells displayed impaired IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking, whereas RapL-KD cells displayed a dramatic increase in IL-2 production upon stimulation under the same conditions. These results indicate that although both RIAM and RapL regulate Rap1-dependent LFA-1 activation, these molecules have distinct roles in regulating signaling and functional outcomes of T cell responses after T cell receptor triggering. Disclosures: No relevant conflicts of interest to declare.
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Weng, Z., S. M. Thomas, R. J. Rickles, J. A. Taylor, A. W. Brauer, C. Seidel-Dugan, W. M. Michael, G. Dreyfuss, and J. S. Brugge. "Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains." Molecular and Cellular Biology 14, no. 7 (July 1994): 4509–21. http://dx.doi.org/10.1128/mcb.14.7.4509-4521.1994.
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Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.
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Weng, Z., S. M. Thomas, R. J. Rickles, J. A. Taylor, A. W. Brauer, C. Seidel-Dugan, W. M. Michael, G. Dreyfuss, and J. S. Brugge. "Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains." Molecular and Cellular Biology 14, no. 7 (July 1994): 4509–21. http://dx.doi.org/10.1128/mcb.14.7.4509.
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Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.
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Foster, R., K. Q. Hu, D. A. Shaywitz, and J. Settleman. "p190 RhoGAP, the major RasGAP-associated protein, binds GTP directly." Molecular and Cellular Biology 14, no. 11 (November 1994): 7173–81. http://dx.doi.org/10.1128/mcb.14.11.7173-7181.1994.
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In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.
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Foster, R., K. Q. Hu, D. A. Shaywitz, and J. Settleman. "p190 RhoGAP, the major RasGAP-associated protein, binds GTP directly." Molecular and Cellular Biology 14, no. 11 (November 1994): 7173–81. http://dx.doi.org/10.1128/mcb.14.11.7173.
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In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.
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Pazman, C., C. A. Mayes, M. Fanto, S. R. Haynes, and M. Mlodzik. "Rasputin, the Drosophila homologue of the RasGAP SH3 binding protein, functions in ras- and Rho-mediated signaling." Development 127, no. 8 (April 15, 2000): 1715–25. http://dx.doi.org/10.1242/dev.127.8.1715.
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The small GTPase Ras plays an important role in many cellular signaling processes. Ras activity is negatively regulated by GTPase activating proteins (GAPs). It has been proposed that RasGAP may also function as an effector of Ras activity. We have identified and characterized the Drosophila homologue of the RasGAP-binding protein G3BP encoded by rasputin (rin). rin mutants are viable and display defects in photoreceptor recruitment and ommatidial polarity in the eye. Mutations in rin/G3BP genetically interact with components of the Ras signaling pathway that function at the level of Ras and above, but not with Raf/MAPK pathway components. These interactions suggest that Rin is required as an effector in Ras signaling during eye development, supporting an effector role for RasGAP. The ommatidial polarity phenotypes of rin are similar to those of RhoA and the polarity genes, e.g. fz and dsh. Although rin/G3BP interacts genetically with RhoA, affecting both photoreceptor differentiation and polarity, it does not interact with the gain-of-function genotypes of fz and dsh. These data suggest that Rin is not a general component of polarity generation, but serves a function specific to Ras and RhoA signaling pathways.
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Weng, Z., R. J. Rickles, S. Feng, S. Richard, A. S. Shaw, S. L. Schreiber, and J. S. Brugge. "Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions." Molecular and Cellular Biology 15, no. 10 (October 1995): 5627–34. http://dx.doi.org/10.1128/mcb.15.10.5627.
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SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands. These results indicate that Asp-99 is important for Src SH3 binding specificity and that Asp-99-dependent binding interactions play a dominant role in Src SH3 recognition of cellular binding proteins, and they suggest the existence of two Src SH3 binding mechanisms, one requiring Asp-99 and the other independent of this residue.
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Becker, Elena, Uyen Huynh-Do, Sacha Holland, Tony Pawson, Tom O. Daniel, and Edward Y. Skolnik. "Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation." Molecular and Cellular Biology 20, no. 5 (March 1, 2000): 1537–45. http://dx.doi.org/10.1128/mcb.20.5.1537-1545.2000.
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ABSTRACT The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62 dok , RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62 dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.
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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.14.9.6372-6385.1994.
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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.
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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.
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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.
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Freeman, N. L., T. Lila, K. A. Mintzer, Z. Chen, A. J. Pahk, R. Ren, D. G. Drubin, and J. Field. "A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization." Molecular and Cellular Biology 16, no. 2 (February 1996): 548–56. http://dx.doi.org/10.1128/mcb.16.2.548.
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Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.
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Bottger, Gina, Phil Barnett, AndréT J. Klein, Astrid Kragt, Henk F. Tabak, and Ben Distel. "Saccharomyces cerevisiae PTS1 Receptor Pex5p Interacts with the SH3 Domain of the Peroxisomal Membrane Protein Pex13p in an Unconventional, Non-PXXP–related Manner." Molecular Biology of the Cell 11, no. 11 (November 2000): 3963–76. http://dx.doi.org/10.1091/mbc.11.11.3963.
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A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p. In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain. Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p. Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way. To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3. Such mutations were all located in a small region in the N-terminal half of Pex5p. One of the altered residues (F208) was part of the sequence W204XXQF208, that is conserved between Pex5 proteins of different species. Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain. The Pex5p mutants could only partially restore PTS1-protein import in pex5Δ cells in vivo. In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3. In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes. Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function.
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Robens, Jeffrey M., Lee Yeow-Fong, Elsa Ng, Christine Hall, and Ed Manser. "Regulation of IRSp53-Dependent Filopodial Dynamics by Antagonism between 14-3-3 Binding and SH3-Mediated Localization." Molecular and Cellular Biology 30, no. 3 (November 23, 2009): 829–44. http://dx.doi.org/10.1128/mcb.01574-08.
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ABSTRACT Filopodia are dynamic structures found at the leading edges of most migrating cells. IRSp53 plays a role in filopodium dynamics by coupling actin elongation with membrane protrusion. IRSp53 is a Cdc42 effector protein that contains an N-terminal inverse-BAR (Bin-amphipysin-Rvs) domain (IRSp53/MIM homology domain [IMD]) and an internal SH3 domain that associates with actin regulatory proteins, including Eps8. We demonstrate that the SH3 domain functions to localize IRSp53 to lamellipodia and that IRSp53 mutated in its SH3 domain fails to induce filopodia. Through SH3 domain-swapping experiments, we show that the related IRTKS SH3 domain is not functional in lamellipodial localization. IRSp53 binds to 14-3-3 after phosphorylation in a region that lies between the CRIB and SH3 domains. This association inhibits binding of the IRSp53 SH3 domain to proteins such as WAVE2 and Eps8 and also prevents Cdc42-GTP interaction. The antagonism is achieved by phosphorylation of two related 14-3-3 binding sites at T340 and T360. In the absence of phosphorylation at these sites, filopodium lifetimes in cells expressing exogenous IRSp53 are extended. Our work does not conform to current views that the inverse-BAR domain or Cdc42 controls IRSp53 localization but provides an alternative model of how IRSp53 is recruited (and released) to carry out its functions at lamellipodia and filopodia.
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Zamanian, Jennifer L., and Regis B. Kelly. "Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis." Molecular Biology of the Cell 14, no. 4 (April 2003): 1624–37. http://dx.doi.org/10.1091/mbc.e02-08-0494.
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Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L.
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Hu, K. Q. "Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation." EMBO Journal 16, no. 3 (February 1, 1997): 473–83. http://dx.doi.org/10.1093/emboj/16.3.473.
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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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Zhao, Zhou-shen, Edward Manser, and Louis Lim. "Interaction between PAK and Nck: a Template for Nck Targets and Role of PAK Autophosphorylation." Molecular and Cellular Biology 20, no. 11 (June 1, 2000): 3906–17. http://dx.doi.org/10.1128/mcb.20.11.3906-3917.2000.
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ABSTRACT The kinase PAK binds tightly to the SH3 domain of its partner PIX via a central proline-rich sequence. A different N-terminal sequence allows αPAK to bind an SH3 domain of the adaptor Nck. The Nck SH3[2] domain interacts equally with an 18-mer PAK-derived peptide and full-length αPAK. Detailed analysis of this binding by saturation substitution allows related Nck targets to be accurately identified from sequence characteristics alone. All Nck SH3[2] binding proteins, including PAK, NIK, synaptojanin, PRK2, and WIP, possess the motif PXXPXRXXS; in the case of PAK, serine phosphorylation at this site negatively regulates binding. We show that kinase autophosphorylation blocks binding by both Nck and PIX to αPAK, thus providing a mechanism to regulate PAK interactions with its SH3-containing partners. One cellular consequence of the regulatable binding of PAK is facilitation of its cycling between cytosolic and focal complex sites.
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Aldehaiman, Abdullah, Afaque A. Momin, Audrey Restouin, Luyao Wang, Xiaoli Shi, Safia Aljedani, Sandrine Opi, et al. "Synergy and allostery in ligand binding by HIV-1 Nef." Biochemical Journal 478, no. 8 (April 21, 2021): 1525–45. http://dx.doi.org/10.1042/bcj20201002.
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The Nef protein of human and simian immunodeficiency viruses boosts viral pathogenicity through its interactions with host cell proteins. By combining the polyvalency of its large unstructured regions with the binding selectivity and strength of its folded core domain, Nef can associate with many different host cell proteins, thereby disrupting their functions. For example, the combination of a linear proline-rich motif and hydrophobic core domain surface allows Nef to bind tightly and specifically to SH3 domains of Src family kinases. We investigated whether the interplay between Nef's flexible regions and its core domain could allosterically influence ligand selection. We found that the flexible regions can associate with the core domain in different ways, producing distinct conformational states that alter the way in which Nef selects for SH3 domains and exposes some of its binding motifs. The ensuing crosstalk between ligands might promote functionally coherent Nef-bound protein ensembles by synergizing certain subsets of ligands while excluding others. We also combined proteomic and bioinformatics analyses to identify human proteins that select SH3 domains in the same way as Nef. We found that only 3% of clones from a whole-human fetal library displayed Nef-like SH3 selectivity. However, in most cases, this selectivity appears to be achieved by a canonical linear interaction rather than by a Nef-like ‘tertiary' interaction. Our analysis supports the contention that Nef's mode of hijacking SH3 domains is a virus-specific adaptation with no or very few cellular counterparts. Thus, the Nef tertiary binding surface is a promising virus-specific drug target.
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Lila, T., and D. G. Drubin. "Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton." Molecular Biology of the Cell 8, no. 2 (February 1997): 367–85. http://dx.doi.org/10.1091/mbc.8.2.367.
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In a variety of organisms, a number of proteins associated with the cortical actin cytoskeleton contain SH3 domains, suggesting that these domains may provide the physical basis for functional interactions among structural and regulatory proteins in the actin cytoskeleton. We present evidence that SH3 domains mediate at least two independent functions of the Saccharomyces cerevisiae actin-binding protein Abp1p in vivo. Abp1p contains a single SH3 domain that has recently been shown to bind in vitro to the adenylyl cyclase-associated protein Srv2p. Immunofluorescence analysis of Srv2p subcellular localization in strains carrying mutations in either ABP1 or SRV2 reveals that the Abp1p SH3 domain mediates the normal association of Srv2p with the cortical actin cytoskeleton. We also show that a site in Abp1p itself is specifically bound by the SH3 domain of the actin-associated protein Rvs167p. Genetic analysis provides evidence that Abp1p and Rvs167p have functions that are closely interrelated. Abp1 null mutations, like rvs167 mutations, result in defects in sporulation and reduced viability under certain suboptimal growth conditions. In addition, mutations in ABP1 and RVS167 yield similar profiles of genetic "synthetic lethal" interactions when combined with mutations in genes encoding other cytoskeletal components. Mutations which specifically disrupt the SH3 domain-mediated interaction between Abp1p and Srv2p, however, show none of the shared phenotypes of abp1 and rvs167 mutations. We conclude that the Abp1p SH3 domain mediates the association of Srv2p with the cortical actin cytoskeleton, and that Abp1p performs a distinct function that is likely to involve binding by the Rvs167p SH3 domain. Overall, work presented here illustrates how SH3 domains can integrate the activities of multiple actin cytoskeleton proteins in response to varying environmental conditions.
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Matsui, Y., R. Matsui, R. Akada, and A. Toh-e. "Yeast src homology region 3 domain-binding proteins involved in bud formation." Journal of Cell Biology 133, no. 4 (May 15, 1996): 865–78. http://dx.doi.org/10.1083/jcb.133.4.865.
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The yeast protein Bem1p, which bears two src homology region 3 (SH3) domains, is involved in cell polarization. A Rho-type GTPase, Rho3p, is involved in the maintenance of cell polarity for bud formation, and the rho3 defect is suppressed by a high dose of BEM1. Mutational analysis revealed that the second SH3 domain from the NH2 terminus (SH3-2) of Bem1p is important for the functions of Bem1p in bud formation and in the suppression of the rho3 defect. Boi2p, which bound to SH3-2 Bem1p, was identified using the two-hybrid system. Boi2p has a proline-rich sequence that is critical for displaying the Boi2p-Bem1p two-hybrid interaction, an SH3 domain in its NH2-terminal half, and a pleckstrin homology domain in its COOH-terminal half. A BOI2 homologue, BOI1, was identified as a gene whose overexpression inhibited cell growth. Cells overexpressing either BOI1 or BOI2 were arrested as large, round, and unbudded cells, indicating that the Boi proteins affect cell polarization. Genetic analysis revealed that BOI1 and BOI2 are functionally redundant and important for cell growth. delta boi1 delta boi2 cells became large round cells or lysed with buds, displaying defects in bud formation and in the maintenance of cell polarity. Analysis using several truncated versions of BOI2 revealed that the COOH-terminal half, which contains the pleckstrin homology domain is essential for the function of Boi2p in cell growth, while the NH2-terminal half is not, and the NH2-terminal half might be required for modulating the function of Bem1p. Overproduction of either Rho3p or the Rho3p-related GTPase Rho4p suppressed the boi defect. These results demonstrate that Rho3p GTPases and Boi proteins function in the maintenance of cell polarity for bud formation.
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RASMUSSEN, Richele K., Jenny RUSAK, Gareth PRICE, Phillip J. ROBINSON, Richard J. SIMPSON, and Donna S. DOROW. "Mixed-lineage kinase 2-SH3 domain binds dynamin and greatly enhances activation of GTPase by phospholipid." Biochemical Journal 335, no. 1 (October 1, 1998): 119–24. http://dx.doi.org/10.1042/bj3350119.
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Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain–glutathione S-transferase fusion protein (GST–MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3´-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin.
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Gigoux, Véronique, Sébastien L'Hoste, Françoise Raynaud, Jacques Camonis, and Christiane Garbay. "Identification of Aurora Kinases as RasGAP Src Homology 3 Domain-binding Proteins." Journal of Biological Chemistry 277, no. 26 (April 25, 2002): 23742–46. http://dx.doi.org/10.1074/jbc.c200121200.
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Eulitz, Stefan, Florian Sauer, Marie-Cecile Pelissier, Prisca Boisguerin, Sibylle Molt, Julia Schuld, Zacharias Orfanos, et al. "Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling." Molecular Biology of the Cell 24, no. 20 (October 15, 2013): 3215–26. http://dx.doi.org/10.1091/mbc.e13-04-0202.
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The Xin actin-binding repeat–containing proteins Xin and XIRP2 are exclusively expressed in striated muscle cells, where they are believed to play an important role in development. In adult muscle, both proteins are concentrated at attachment sites of myofibrils to the membrane. In contrast, during development they are localized to immature myofibrils together with their binding partner, filamin C, indicating an involvement of both proteins in myofibril assembly. We identify the SH3 domains of nebulin and nebulette as novel ligands of proline-rich regions of Xin and XIRP2. Precise binding motifs are mapped and shown to bind both SH3 domains with micromolar affinity. Cocrystallization of the nebulette SH3 domain with the interacting XIRP2 peptide PPPTLPKPKLPKH reveals selective interactions that conform to class II SH3 domain–binding peptides. Bimolecular fluorescence complementation experiments in cultured muscle cells indicate a temporally restricted interaction of Xin-repeat proteins with nebulin/nebulette during early stages of myofibril development that is lost upon further maturation. In mature myofibrils, this interaction is limited to longitudinally oriented structures associated with myofibril development and remodeling. These data provide new insights into the role of Xin actin-binding repeat–containing proteins (together with their interaction partners) in myofibril assembly and after muscle damage.
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Jia, Xuanyan, Leishu Lin, Shun Xu, Lingxuan Li, Zhiyi Wei, Cong Yu, and Fengfeng Niu. "Crystal Structure of the SH3 Domain of ASAP1 in Complex with the Proline Rich Motif (PRM) of MICAL1 Reveals a Unique SH3/PRM Interaction Mode." International Journal of Molecular Sciences 24, no. 2 (January 11, 2023): 1414. http://dx.doi.org/10.3390/ijms24021414.
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SH3 domains are common protein binding modules. The target sequence of SH3 domains is usually a proline-rich motif (PRM) containing a minimal “PxxP” sequence. The mechanism of how different SH3 domains specifically choose their targets from vast PxxP-containing sequences is still not very clear, as many reported SH3/PRM interactions are weak and promiscuous. Here, we identified the binding of the SH3 domain of ASAP1 to the PRM of MICAL1 with a sub-μM binding affinity, and determined the crystal structure of ASAP1-SH3 and MICAL1-PRM complex. Our structural and biochemical analyses revealed that the target-binding pocket of ASAP1-SH3 contains two negatively charged patches to recognize the “xPx + Px+” sequence in MICAL1-PRM and consequently strengthen the interaction, differing from the typical SH3/PRM interaction. This unique PRM-binding pocket is also found in the SH3 domains of GTPase Regulator associated with focal adhesion kinase (GRAF) and Src kinase associated phosphoprotein 1 (SKAP1), which we named SH3AGS. In addition, we searched the Swiss-Prot database and found ~130 proteins with the SH3AGS-binding PRM in silico. Finally, gene ontology analysis suggests that the strong interaction between the SH3AGS-containing proteins and their targets may play roles in actin cytoskeleton regulation and vesicle trafficking.
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OHBA, Takeaki, Masaho ISHINO, Hiroshi AOTO, and Terukatsu SASAKI. "Interaction of two proline-rich sequences of cell adhesion kinase β with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf." Biochemical Journal 330, no. 3 (March 15, 1998): 1249–54. http://dx.doi.org/10.1042/bj3301249.
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Cell adhesion kinase β (CAKβ) is a protein tyrosine kinase closely related to focal adhesion kinase (FAK) in structure. CAKβ contains two proline-rich sequences within its C-terminal region. Since proline-rich sequences present in the corresponding region of FAK are known to mediate protein-protein interactions by binding to SH3 domains, we investigated binding of CAKβ to a panel of SH3 domains. Affinity precipitation from rat brain lysate revealed selective interactions of CAKβ with glutathione S-transferase (GST)-fused SH3 domains of p130Cas(Cas)-related proteins and Graf. Mutational analysis indicated that the proline-rich sequences of CAKβ mediate this interaction. Each of the two proline-rich sequences fused to GST bound directly to these SH3 domains in dot blot analysis. A competitive binding assay revealed that the first proline-rich sequence of CAKβ preferentially associated with the SH3 domain of Cas. The second proline-rich sequence of CAKβ bound to the SH3 domain of Graf with higher specificity than the corresponding proline-rich sequence of FAK. Finally, we showed co-immunoprecipitation of CAKβ with Graf from rat brain lysate. These results indicate that CAKβ associates in vivo with Graf through its SH3 domain.
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Murayama, Kazutaka, Mikako Shirouzu, Yoshihiro Kawasaki, Miyuki Kato-Murayama, Kyoko Hanawa-Suetsugu, Ayako Sakamoto, Yasuhiro Katsura, et al. "Crystal Structure of the Rac Activator, Asef, Reveals Its Autoinhibitory Mechanism." Journal of Biological Chemistry 282, no. 7 (December 26, 2006): 4238–42. http://dx.doi.org/10.1074/jbc.c600234200.
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The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.
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Li, Bo, Lei Zhuang, and Beat Trueb. "Zyxin Interacts with the SH3 Domains of the Cytoskeletal Proteins LIM-nebulette and Lasp-1." Journal of Biological Chemistry 279, no. 19 (March 5, 2004): 20401–10. http://dx.doi.org/10.1074/jbc.m310304200.
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Zyxin is a versatile component of focal adhesions in eukaryotic cells. Here we describe a novel binding partner of zyxin, which we have named LIM-nebulette. LIM-nebulette is an alternative splice variant of the sarcomeric protein nebulette, which, in contrast to nebulette, is expressed in non-muscle cells. It displays a modular structure with an N-terminal LIM domain, three nebulin-like repeats, and a C-terminal SH3 domain and shows high similarity to another cytoskeletal protein, Lasp-1 (LIM and SH3 protein-1). Co-precipitation studies and results obtained with the two-hybrid system demonstrate that LIM-nebulette and Lasp-1 interact specifically with zyxin. Moreover, the SH3 domain from LIM-nebulette is both necessary and sufficient for zyxin binding. The SH3 domains from Lasp-1 and nebulin can also interact with zyxin, but the SH3 domains from more distantly related proteins such as vinexin and sorting nexin 9 do not. On the other hand, the binding site in zyxin is situated at the extreme N terminus as shown by site-directed mutagenesis. LIM-nebulette and Lasp-1 use the same linear binding motif. This motif shows some similarity to a class II binding site but does not contain the classical PXXP sequence. LIM-nebulette reveals a subcellular distribution at focal adhesions similar to Lasp-1. Thus, LIM-nebulette, Lasp-1, and zyxin may play an important role in the organization of focal adhesions.
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Zhu, J., and S. K. Shore. "c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein." Molecular and Cellular Biology 16, no. 12 (December 1996): 7054–62. http://dx.doi.org/10.1128/mcb.16.12.7054.
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The c-ABL tyrosine kinase is activated following either the loss or mutation of its Src homology domain 3 (SH3), resulting in both increased autophosphorylation and phosphorylation of cellular substrates and cellular transformation. This suggests that the SH3 domain negatively regulates c-ABL kinase activity. For several reasons this regulation is thought to involve a cellular protein that binds to the SH3 domain. Hyperexpression of c-ABL results in an activation of its kinase, the kinase activity of purified c-ABL protein in the absence of cellular proteins is independent of either the presence or absence of a SH3 domain, and point mutations and deletions within the SH3 domain are sufficient to activate c-ABL transforming ability. To identify proteins that interact with the c-ABL SH3 domain, we screened a cDNA library by the yeast two-hybrid system, using the c-ABL SH3SH2 domains as bait. We identified a novel protein, AAP1 (ABL-associated protein 1), that associates with these c-ABL domains and fails to bind to the SH3 domain in the activated oncoprotein BCRABL. Kinase experiments demonstrated that in the presence of AAP1, the ability of c-ABL to phosphorylate either glutathione S-transferase-CRK or enolase was inhibited. In contrast, AAP1 had little effect on the phosphorylation of glutathione S-transferase-CRK by the activated ABL oncoproteins v-ABL and BCRABL. We conclude that AAP1 inhibits c-ABL tyrosine kinase activity but has little effect on the tyrosine kinase activities of oncogenic BCRABL or v-ABL protein and propose that AAP1 functions as a trans regulator of c-ABL kinase. Our data also indicate that loss of susceptibility to AAP1 regulation correlates with oncogenicity of the activated forms of c-ABL.
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Norton, Robert, Zhi Huang, Jeffrey Fay, Fukun Hoffmann, and Peter Hoffmann. "Selenoprotein K interacts with ASAP2 in macrophages to promote phagocytosis (P4151)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 112.9. http://dx.doi.org/10.4049/jimmunol.190.supp.112.9.
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Abstract Selenium is a mineral micronutrient that is essential in a variety of biological systems, including immune responses. Selenium is used for biosynthesis of the amino acid selenocysteine, which is used by selenoproteins to carry out biological functions. In humans, 25 selenoproteins have been identified and Selenoprotein K (SelK) was recently identified as an endoplasmic reticulum transmembrane protein important for calcium flux during the activation of immune cells. ASAP2 is an ARF-GAP that localizes to the phagocytic cup when a macrophage engulfs a pathogen. Implementation of the SH3 hunter software predicted that the SH3-binding domain of SelK associates with SH3 domain containing proteins. Furthermore, in an unbiased screen for SelK binding partners we found that ASAP2 immunoprecipitates with SelK. When we tested mutants of SelK with alterations in the SH3 binding domain, we discovered that this interaction was abolished. This indicates that SelK has a functional SH3 binding domain that interacts directly with ASAP2. It has also been established by our lab that SelK KO macrophages exhibit notably impaired phagocytosis of opsonized beads. Studies are underway to determine if the lack of SelK prevents ASAP2 from localizing to the phagocytic cup.
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Risse, Sarah L., Belen Vaz, Matthew F. Burton, Pontus Aspenström, Roland P. Piekorz, Luc Brunsveld, and Mohammad R. Ahmadian. "SH3-mediated targeting of Wrch1/RhoU by multiple adaptor proteins." Biological Chemistry 394, no. 3 (March 1, 2013): 421–32. http://dx.doi.org/10.1515/hsz-2012-0246.
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Abstract Wrch1/RhoU is an atypical member of the Rho family. A major structural difference is the extended N-terminus of Wrch1 (nWrch1) containing three putative SH3 domain-binding motifs whose specificities are unknown. To define the impact of this extended region on coupling Wrch1 to cellular signaling, we analyzed in this study nWrch1 interaction with Src homology 3 (SH3) domains of different adaptor proteins. Using sedimentation and isothermal titration calorimetric (ITC) measurements, we identified isolated SH3 domains of growth factor receptor-bound protein 2 (Grb2), noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), c-Src, chicken tumor virus no. 10 (CT 10) regulator kinase 1 (Crk1), and p120 as low-affinity Wrch1-binding partners. Interestingly, under cell-based conditions, nWrch1 bound tightly to endogenous Grb2 and Nck, but not to Crk, c-Src, or p120. Consistent with this, a very tight nWrch1 interaction with full-length Grb2 and Nck1 was confirmed in vitro by ITC measurements indicating that high avidity of the adaptor proteins can compensate for the low affinity of their SH3 domains. Peptide analysis revealed that the central PxxP motif of nWrch1, which employs a minimal consensus sequence of eight amino acids with an essential arginine next to the PxxP motif, is responsible for these interactions. Thus, novel functional insights from this study suggest that multiple upstream signals may converge on Wrch1 directly through its SH3 domain-binding properties.
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Lefferts, Joel A., Chuan Wang, Melissa Baralt, and Muriel W. Lambert. "The Fanconi Anemia Protein, FANCG, Binds Directly to the SH3 Domain of aII Spectrin and This Interaction May Play An Important Role in the Stability of aII Spectrin and Its Function in Repair of DNA Interstrand Cross-Links." Blood 112, no. 11 (November 16, 2008): 1041. http://dx.doi.org/10.1182/blood.v112.11.1041.1041.
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Abstract Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, a predisposition to cancer, congenital abnormalities and a cellular hypersensitivity to DNA interstrand cross-linking agents. This hypersensitivity in FA cells correlates with a defect in ability to repair interstrand cross-links. We have shown that the structural protein, nonerythroid a spectrin (aIISp), plays an important role in the repair of DNA interstrand cross-links and that there is a deficiency in aIISp in FA cells. The reduced levels of aIISp in FA cells are due to reduced stability of this protein. We propose that the stability of aIISp is dependent upon one or more of the FA proteins. The present study was undertaken in order to get a clearer understanding of the proposed role of FA proteins in maintaining the stability of aIISp in the cell and the functional importance of this relationship in the repair of DNA interstrand cross-links and the repair defect in FA cells. For these studies, FA proteins were examined for their ability to directly interact with aIISp and, if so, to map the sites of interaction. Four overlapping regions of aIISp were constructed and binding of FA proteins to each of these regions was examined using yeast two-hybrid analysis. Of the proteins examined, FANCG was found to interact with one of these regions of aIISp and specifically with the SH3 domain within this region. The site of interaction in FANCG was mapped to a SH3 domain binding motif, which contains a consensus sequence with preference for the SH3 domain of aIISp. The sites of interaction in both FANCG and aIISp were confirmed using site-directed mutagenesis. Two FA proteins that did not contain any SH3 binding motifs, FANCC and FANCF, did not interact with the SH3 domain of aIISp or any of the other regions of aIISp. These results thus demonstrate that one of the FA proteins, FANCG, contains a class of motifs that has specificity for binding to SH3 domains and binds to the SH3 domain of aIISp via this motif. This binding is important in the DNA repair process and in the FA repair defect, as is shown by our in vivo studies in which FA-G cells, transformed with the FANCG cDNA and stably expressing FANCG, showed normal levels of aIISp and a correction of the defect in ability to repair DNA interstrand cross-links. We therefore propose that the binding of FANCG to aIISp is not only important for the stability of aIISp in cells but also for the role aIISp plays in the DNA repair process. Thus a deficiency in FANCG, such as occurs in FA-G cells, could lead to reduced stability of aIISp which in turn could be an important factor in the defective DNA repair pathway in FA cells.
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Hiipakka, Marita, and Kalle Saksela. "Capacity of simian immunodeficiency virus strain mac Nef for high-affinity Src homology 3 (SH3) binding revealed by ligand-tailored SH3 domains." Journal of General Virology 83, no. 12 (December 1, 2002): 3147–52. http://dx.doi.org/10.1099/0022-1317-83-12-3147.
Full textAbstract:
The simian immunodeficiency virus (SIV) Nef protein contains a consensus Src-homology 3 (SH3) binding motif. However, no SH3-domain proteins showing strong binding to SIV Nef have yet been found, and its potential capacity for high-affinity SH3 binding has therefore remained unproven. Here we have used phage-display-assisted protein engineering to develop artificial SH3 domains that bind tightly to SIV strain mac (SIVmac) Nef. Substitution of six amino acids in the RT loop region of Hck-SH3 with the sequence E/DGWWG resulted in SH3 domains that bound in vitro to SIVmac Nef much better than the natural Hck- or Fyn-SH3 domains. These novel SH3 domains also efficiently associated with SIVmac Nef when co-expressed in 293T cells and displayed a strikingly differential specificity when compared with SH3 domains similarly targeted for binding to human immunodeficiency virus type 1 (HIV-1) Nef. Thus, SIVmac Nef is competent for high-affinity SH3 binding, but its natural SH3 protein partners are likely to be different from those of HIV-1 Nef.
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Parker, F., F. Maurier, I. Delumeau, M. Duchesne, D. Faucher, L. Debussche, A. Dugue, F. Schweighoffer, and B. Tocque. "A Ras-GTPase-activating protein SH3-domain-binding protein." Molecular and Cellular Biology 16, no. 6 (June 1996): 2561–69. http://dx.doi.org/10.1128/mcb.16.6.2561.
Full textAbstract:
We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.
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Mayer, B. J. "SH3 domains: complexity in moderation." Journal of Cell Science 114, no. 7 (April 1, 2001): 1253–63. http://dx.doi.org/10.1242/jcs.114.7.1253.
Full textAbstract:
The SH3 domain is perhaps the best-characterized member of the growing family of protein-interaction modules. By binding with moderate affinity and selectivity to proline-rich ligands, these domains play critical roles in a wide variety of biological processes ranging from regulation of enzymes by intramolecular interactions, increasing the local concentration or altering the subcellular localization of components of signaling pathways, and mediating the assembly of large multiprotein complexes. SH3 domains and their binding sites have cropped up in many hundreds of proteins in species from yeast to man, which suggests that they provide the cell with an especially handy and adaptable means of bringing proteins together. The wealth of genetic, biochemical and structural information available provides an intimate and detailed portrait of the domain, serving as a framework for understanding other modular protein-interaction domains. Processes regulated by SH3 domains also raise important questions about the nature of specificity and the overall logic governing networks of protein interactions.
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Bustelo, X. R., K. L. Suen, W. M. Michael, G. Dreyfuss, and M. Barbacid. "Association of the vav proto-oncogene product with poly(rC)-specific RNA-binding proteins." Molecular and Cellular Biology 15, no. 3 (March 1995): 1324–32. http://dx.doi.org/10.1128/mcb.15.3.1324.
Full textAbstract:
We have used the yeast two-hybrid system to isolate proteins that interact with the carboxy-terminal SH3-SH2-SH3 region of Vav. One of the clones encoded heterogeneous nuclear ribonucleoprotein K (hnRNP K), a poly(rC)-specific RNA-binding protein. The interaction between Vav and hnRNP K involves the binding of the most carboxy-terminal SH3 domain of Vav to two proline-rich sequences present in the central region of hnRNP K. Overexpression of Vav in mouse fibroblasts leads to the formation of a stable complex with the endogenous hnRNP K and to the preferential redistribution of this protein to the cytoplasmic fraction. More importantly, Vav and hnRNP K proteins also interact in hematopoietic cells. In addition, Vav associates in vitro with a second 45-kDa poly(rC)-specific RNA-binding protein via its SH3-SH2-SH3 region. These results suggest that Vav plays a role in the regulation of the late steps of RNA biogenesis by modulating the function of poly(rC)-specific ribonucleoproteins.
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Tsapara, Anna, Karl Matter, and Maria S. Balda. "The Heat-Shock Protein Apg-2 Binds to the Tight Junction Protein ZO-1 and Regulates Transcriptional Activity of ZONAB." Molecular Biology of the Cell 17, no. 3 (March 2006): 1322–30. http://dx.doi.org/10.1091/mbc.e05-06-0507.
Full textAbstract:
The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1–ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G1/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1–ZONAB signaling in epithelial cells in response to cellular stress.
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Nakamoto, T., R. Sakai, H. Honda, S. Ogawa, H. Ueno, T. Suzuki, S. Aizawa, Y. Yazaki, and H. Hirai. "Requirements for localization of p130cas to focal adhesions." Molecular and Cellular Biology 17, no. 7 (July 1997): 3884–97. http://dx.doi.org/10.1128/mcb.17.7.3884.
Full textAbstract:
p130cas (Cas) is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respectively. Cas was originally identified as a major tyrosine-phosphorylated protein in v-Crk- and v-Src-transformed cells. Subsequently, Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation; it is therefore regarded as one of the focal adhesion proteins. Using an immunofluorescence study, we examined the subcellular localization of Cas and determined the regions required for its localization to focal adhesions. In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions. However, in 527F-c-Src-transformed cells, Cas was localized mainly to podosomes, where the focal adhesion proteins are assembled. The localization of Cas to focal adhesions was also observed in cells expressing the kinase-negative 527F/295M-c-Src. A series of analyses with deletion mutants expressed in various cells revealed that the SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transformed cells and fibronectin-stimulated cells. In addition, the localization of Cas to focal adhesions was abolished in Src-negative cells. These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Cas to focal adhesions.
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DeJournett, Robert E., Ryuji Kobayashi, Shujuan Pan, Chuanfen Wu, Laurence D. Etkin, Richard B. Clark, Oliver Bögler, and Jian Kuang. "Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins." Biochemical Journal 401, no. 2 (December 21, 2006): 521–31. http://dx.doi.org/10.1042/bj20061287.
Full textAbstract:
The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (α-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.
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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.
Full textAbstract:
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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Pietrek, Marcel, Melanie M. Brinkmann, Ilona Glowacka, Anette Enlund, Anika Hävemeier, Oliver Dittrich-Breiholz, Michael Kracht, et al. "Role of the Kaposi's Sarcoma-Associated Herpesvirus K15 SH3 Binding Site in Inflammatory Signaling and B-Cell Activation." Journal of Virology 84, no. 16 (June 9, 2010): 8231–40. http://dx.doi.org/10.1128/jvi.01696-09.
Full textAbstract:
ABSTRACT The Kaposi's sarcoma-associated herpesvirus (KSHV) contains several open reading frames (ORFs) that encode proteins capable of initiating and modulating cellular signaling pathways. Among them is ORF K15, encoding a 12-transmembrane-spanning protein with a cytoplasmic C-terminal domain. Through conserved binding motifs, such as Src homology 2 (SH2) and SH3 binding sites, K15 interacts with cellular proteins, activates the NF-κB, MEK/Erk, and Jun N-terminal protein kinase (JNK) pathways, and induces the expression of several inflammatory and angiogenic genes. In this study, we investigated the role of an SH3 domain binding site centered on a PPLP motif in K15. We screened libraries of cellular SH3 domains to identify signaling molecules interacting with the KSHV PPLP motif. We found its affinities for two Src kinase family members, Lyn and Hck, to exceed those of other viral proteins. While the SH2 binding motif YEEV is essential for the inflammatory response induced by KSHV K15, recruitment of Lyn and Hck to the K15 PPLP motif seems to be dispensable for this inflammatory response. However, the PPLP motif is essential for the decrease in B-cell receptor-mediated signaling induced by K15, as measured by calcium mobilization assays.
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Zhao, Zhe, Riku Fagerlund, Helena Tossavainen, Kristina Hopfensperger, Rishikesh Lotke, Smitha Srinivasachar Badarinarayan, Frank Kirchhoff, et al. "Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage." PLOS Pathogens 17, no. 11 (November 15, 2021): e1009728. http://dx.doi.org/10.1371/journal.ppat.1009728.
Full textAbstract:
The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of R-clamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.
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Tomida, Seita, Misaki Takata, Tetsuya Hirata, Masamichi Nagae, Miyako Nakano, and Yasuhiko Kizuka. "The SH3 domain in the fucosyltransferase FUT8 controls FUT8 activity and localization and is essential for core fucosylation." Journal of Biological Chemistry 295, no. 23 (April 29, 2020): 7992–8004. http://dx.doi.org/10.1074/jbc.ra120.013079.
Full textAbstract:
Core fucose is an N-glycan structure synthesized by α1,6-fucosyltransferase 8 (FUT8) localized to the Golgi apparatus and critically regulates the functions of various glycoproteins. However, how FUT8 activity is regulated in cells remains largely unclear. At the luminal side and uncommon for Golgi proteins, FUT8 has an Src homology 3 (SH3) domain, which is usually found in cytosolic signal transduction molecules and generally mediates protein-protein interactions in the cytosol. However, the SH3 domain has not been identified in other glycosyltransferases, suggesting that FUT8's functions are selectively regulated by this domain. In this study, using truncated FUT8 constructs, immunofluorescence staining, FACS analysis, cell-surface biotinylation, proteomics, and LC-electrospray ionization MS analyses, we reveal that the SH3 domain is essential for FUT8 activity both in cells and in vitro and identified His-535 in the SH3 domain as the critical residue for enzymatic activity of FUT8. Furthermore, we found that although FUT8 is mainly localized to the Golgi, it also partially localizes to the cell surface in an SH3-dependent manner, indicating that the SH3 domain is also involved in FUT8 trafficking. Finally, we identified ribophorin I (RPN1), a subunit of the oligosaccharyltransferase complex, as an SH3-dependent binding protein of FUT8. RPN1 knockdown decreased both FUT8 activity and core fucose levels, indicating that RPN1 stimulates FUT8 activity. Our findings indicate that the SH3 domain critically controls FUT8 catalytic activity and localization and is required for binding by RPN1, which promotes FUT8 activity and core fucosylation.
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Brown, Megan T., Josefa Andrade, Harish Radhakrishna, Julie G. Donaldson, Jonathan A. Cooper, and Paul A. Randazzo. "ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src." Molecular and Cellular Biology 18, no. 12 (December 1, 1998): 7038–51. http://dx.doi.org/10.1128/mcb.18.12.7038.
Full textAbstract:
ABSTRACT Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.
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Oda, Atsushi, Hans D. Ochs, Laurence A. Lasky, Susan Spencer, Katsutoshi Ozaki, Mitsuhiro Fujihara, Makoto Handa, Kenji Ikebuchi, and Hisami Ikeda. "CrkL is an adapter for Wiskott-Aldrich syndrome protein and Syk." Blood 97, no. 9 (May 1, 2001): 2633–39. http://dx.doi.org/10.1182/blood.v97.9.2633.
Full textAbstract:
Abstract Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia are caused by mutations of the WAS protein (WASP) gene. WASP may be involved in the regulation of podosome, an actin-rich dynamic cell adhesion structure formed by various types of cells. The molecular links between WASP and podosomes or other cell adhesion structures are unknown. Platelets express an SH2-SH3 adapter molecule, CrkL, that can directly associate with paxillin, which is localized in podosomes. The hypothesis that CrkL binds to WASP was, therefore, tested. Results from coprecipitation experiments using anti-CrkL and GST-fusion proteins suggest that CrkL binds to WASP through its SH3 domain and that the binding was not affected by WASP tyrosine phosphorylation. The binding of GST-fusion SH3 domain of PSTPIP1 in vitro was also not affected by WASP tyrosine phosphorylation, suggesting that the binding of the SH3 domains to WASP is not inhibited by tyrosine phosphorylation of WASP. Anti-CrkL also coprecipitates a 72-kd protein, which was identified as syk tyrosine kinase, critical for collagen induced-platelet activation. CrkL immunoprecipitates contain kinase-active syk, as evidenced by an in vitro kinase assay. Coprecipitation experiments using GST-fusion CrkL proteins suggest that both SH2 and SH3 domains of CrkL are involved in the binding of CrkL to syk. WASP, CrkL, syk, and paxillin-like Hic-5 incorporated to platelet cytoskeleton after platelet aggregation. Thus, CrkL is a novel molecular adapter for WASP and syk and may potentially transfer these molecules to the cytoskeleton through association with cytoskeletal proteins such as Hic-5.
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Merilainen, J., R. Palovuori, R. Sormunen, V. M. Wasenius, and V. P. Lehto. "Binding of the alpha-fodrin SH3 domain to the leading lamellae of locomoting chicken fibroblasts." Journal of Cell Science 105, no. 3 (July 1, 1993): 647–54. http://dx.doi.org/10.1242/jcs.105.3.647.
Full textAbstract:
Fodrin (nonerythroid spectrin) is a membrane skeletal protein that plays an important role in the establishment and maintenance of the cell shape and polarity. We have identified in alpha-fodrin an src homology 3 (SH3)-related region, a small domain that is present in a large number of proteins that are involved in signal transduction, cell polarization and membrane-cytoskeleton interactions. In this study we have explored the function of the alpha-fodrin SH3 by incubating fixed and permeabilized cultured chicken fibroblasts with the alpha-fodrin SH3 peptide, expressed in bacteria as a fusion protein with glutathione S-transferase. Immunofluorescence and immunoelectron microscopy showed that alpha-fodrin SH3 binds to the cytoplasmic face of the plasma membrane in the leading lamellae and the pseudopodial lobes of the spreading and locomoting cells. No, or only minimal, binding was seen in immotile cells, or in the stationary trailing ends of the locomoting cells. SH3 binding was also seen in cytochalasin-D-treated cells, suggesting that actin filaments are not responsible for the binding. These findings suggest that alpha-fodrin SH3 interacts with plasma membrane components that are present in the leading lamellae exclusively or are modulated in a manner specific to the leading lamellae.
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Pagano, Livia, Francesca Malagrinò, Caterina Nardella, Stefano Gianni, and Angelo Toto. "Experimental Characterization of the Interaction between the N-Terminal SH3 Domain of Crkl and C3G." International Journal of Molecular Sciences 22, no. 24 (December 7, 2021): 13174. http://dx.doi.org/10.3390/ijms222413174.
Full textAbstract:
Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein–protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G (also known as RAPGEF1), an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains.