Catalytic Activity of the SH2 Domain of Human pp60c-src; Evidence from NMR, Mass Spectrometry, Site-Directed Mutagenesis and Kinetic Studies for an Inherent Phosphatase Activity

Rj, Boerner; Tg, Consler; Rt, Gampe; Weigl, Debra; Dh, Willard; Dg, Davis; Am, Edison; Loganzo, Frank; Db, Kassel; Rx, Xu

doi:10.1021/bi00046a044

Cited by 11 publications

(7 citation statements)

References 30 publications

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“…In addition, the Cys42 side chain has been associated with a weak phosphoesterase activity that may serve to regulate the binding affinity or timescale of interaction between phosphopeptides and the Src SH2 domain 62. The increased preference for the thiol to be deprotonated in the context of the pY‐binding site, manifested in the ∼0.5–0.8 pH unit decrease in p K a compared to solvent‐exposed cysteine (p K a ∼ 9.3), might explain the reported increased catalytic activity of this thiol at physiological pH.…”

Section: Resultsmentioning

confidence: 99%

Structure, dynamics, and binding thermodynamics of the v‐Src SH2 domain: Implications for drug design

et al. 2008

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SH2 domains provide fundamental recognition sites in tyrosine kinase-mediated signaling pathways which, when aberrant, give rise to disease states such as cancer, diabetes, and immune deficiency. Designing specific inhibitors that target the SH2 domain-binding site, however, have presented a major challenge. Despite well over a decade of intensive research, clinically useful SH2 domain inhibitors have yet to become available. A better understanding of the structural, dynamic, and thermodynamic contributions to ligand binding of individual SH2 domains will provide some insight as to whether inhibitor development is possible. We report the first high resolution solution structure of the apo-v-Src SH2 domain. This is accompanied by the analysis of backbone dynamics and pK(a) values within the apo- and peptide-bound states. Our results indicate that the phosphotyrosine (pY) pocket is tightly structured and hence not adaptable to exogenous ligands. On the other hand, the pocket which accommodates residues proximal and C-terminal of the pY (pY + 3) or so-called specificity determining region, is a large dynamic-binding surface. This appears to allow a high level of promiscuity in binding. Binding of a series of synthetic, phosphotyrosyl, peptidomimetic compounds designed to explore interactions in the pY + 3 pocket further demonstrates the ability of the SH2 domain to accommodate diverse ligands. The thermodynamic parameters of these interactions show dramatic enthalpy/entropy compensation. These data suggest that the v-Src SH2 domain does not have a highly specific secondary-binding site, which clearly presents a major hurdle to design selective inhibitors.

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Section: Resultsmentioning

confidence: 99%

Structure, dynamics, and binding thermodynamics of the v‐Src SH2 domain: Implications for drug design

et al. 2008

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“…Peptides were immobilized on a sensor chip by covalent coupling to a carboxylated dextran matrix on its surface as described (34). Purified recombinant Src SH2 and SH3/SH2 domains were expressed and purified as described (35,36) and injected over the sensor chips in 10 mM HEPES pH 7.4, 150 mM NaCl, and 0.05% (v/v) of a 10% P-20 surfactant solution. As described (34) data points were collected when the responses reached steady state and the equilibrium dissociation constants calculated using the Hill equation,…”

Section: Methodsmentioning

confidence: 99%

SH2- and SH3-mediated Interactions between Focal Adhesion Kinase and Src

Thomas

Ellis

Boerner

et al. 1998

Journal of Biological Chemistry

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226

189

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Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. We show that a consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2-and SH3-binding sites exhibits increased affinity for Src. Furthermore, the presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAK Pro-2 ) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo.The x-ray crystal structures of Src and Hck (a Src family member) in their inactive form have revealed intramolecular interactions that function to regulate these protein-tyrosine kinases (PTKs) 1 (1, 2). As expected, the tyrosine phosphorylated, negative regulatory element binds to the SH2 domain (1, 2). In addition, the SH3 domain binds to a polypeptide linking the SH2 and catalytic domains which assumes a polyproline type II helix that is structurally similar to SH3-binding sites (3)(4)(5). In this conformation, ␣-helix C in the small lobe of the catalytic domain is displaced altering the conformation of the ATP-binding site. It is noteworthy that the sequences that bind the SH2 and SH3 domains do not conform to high affinity binding sites (3, 6 -8).Dephosphorylation of the negative regulatory element is one mechanism by which these PTKs can be activated. Consequently, this element fails to bind the SH2 domain and the inactive conformation cannot be maintained. A second mechanism by which the Src-like kinases could be activated is by the disruption of the weaker intramolecular SH2-SH3 interactions by the formation of stronger intermolecular SH2-SH3 domain interactions. It has been shown that disruption of the SH2-or SH3-mediated intramolecular interactions in vitro enhances the activity of the enzyme (9 -12). It is likely that similar mechanisms operate in vivo and that complex formation between Src and its binding partners, like FAK, results in its activation. FAK is a 125-kDa PTK that localizes to focal adhesions and functions in integrin signaling (13,14). Integrin-dependent cell adhesion or cross-linking of cell surface integrins induces the tyrosine phosphorylation of FAK and stimulates its activity (14 -18). The major site of FAK autophosphorylation is Tyr-397, whereas other sites of FAK phosphorylation, e.g. Tyr-576, -577, and -925, are phosphorylated by Src family PTKs (19 -21). The sequence flanking Tyr-397 conforms to a high affinity binding site for the Src SH2 domain and serves as a bind...

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“…Off-line sampling enables the inclusion of any suitable treatment or separation technique necessary to eliminate the possible sources of interference, or to prepare sample solutions for analysis by any available ionization method. For this reason, off-line applications have been accomplished by using a variety of techniques, including FAB/CF-FAB (Caprioli, 1985(Caprioli, , 1988aShao et al, 1987;Langridge et al, 1993;Newton et al, 1997); ESI-MS (Wilson, Perez, & Pasternak, 1993;Aliprandis & Canary, 1994;Gonnet et al, 1996;Charbonniere et al, 1997;Takayama et al, 1997;Wu et al, 1997;Esperson et al, 1998;Kelleher, Hendrickson, & Walsh, 1999;Bothner et al, 2000;Fabris, 2000;Ge et al, 2001;Norris et al, 2001a,b;Pi et al, 2002); LC-MS (Boerner et al, 1995;Hsieh et al, 1995;Li et al, 2000); and MALDI (Craig et al, 1994;Matsumoto, Kahn, & Komori, 1998).…”

Section: Off-line Samplingmentioning

confidence: 99%

“…These advances have enabled the direct characterization of stable covalent adducts formed by many classes of enzymes with substrates, intermediates, products, or inhibitors (Aplin et al, 1990;Stevenson, Feng, & Storer, 1990;Ashton et al, 1991;Menard et al, 1991;Shneier et al, 1991;Orning et al, 1992;Knight et al, 1993;Miao et al, 1994;Boerner et al, 1995;Nairn et al, 1995;Saves et al, 1995;Boerner et al, 1996;Brown, Aplin, & Schofield, 1996;Thiruvikraman et al, 1996;Branchini et al, 1997;Febbraio et al, 1997;Gigant et al, 1997;Ramjee et al, 1997;Skorey et al, 1997;Wandall et al, 1997;Srivastava et al, 1999;Wu et al, 1999;Ichiyama et al, 2000;Regal et al, 2000;Vocadlo et al, 2001). Furthermore, since the initial work reporting the observation of an intact Michaelis complex between hen egg white lysozyme and the hexasaccharide substrate N-acetylglucosamine (NAG 6 ) (Ganem, Li, & Henion, 1991), ESI-MS has been widely used to obtain the stoichiometry, composition, and binding strength of non-covalent complexes formed by the interaction of proteins with different ligands (substrates, products, inhibitors, cofactors, etc.).…”

Section: Off-line Investigation Of Catalysis In Biological Systemsmentioning

confidence: 99%

Mass spectrometric approaches for the investigation of dynamic processes in condensed phase

Fabris

2004

Mass Spectrometry Reviews

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Mass spectrometry (MS) offers many advantages over other established spectroscopic techniques employed for the investigation of processes in condensed phase. The sensitivity, specificity, and speed afforded by MS-based methods enable to obtain very valuable insights into the mechanism of complex dynamic processes. Off-line methods rely on quenching to halt the progress of the reaction of interest and allow for the implementation of a broad range of analytical procedures for sample fractionation, isolation, or desalting. On the contrary, on-line methods are designed to carry out the real-time monitoring of dynamic processes through a continuous uninterrupted analysis of reaction mixtures, with the only caveat that the sample solutions be directly amenable to the available ionization technique. The utilization of rapid mixing devices in direct connection with a mass spectrometer or included in off-line schemes provides access to the initial moments of a reaction, which can offer very important information about the reaction mechanism. This report summarizes the different off- and on-line strategies developed to study chemical and biochemical reactions in solution and obtain kinetic/mechanistic information. The merits of the various experimental designs, the characteristics of the different instrumental setups, and the factors affecting time resolution are discussed with the aid of specific examples, which highlight the contributions of MS to the different facets of the investigation of dynamic processes in condensed phase.

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Catalytic Activity of the SH2 Domain of Human pp60c-src; Evidence from NMR, Mass Spectrometry, Site-Directed Mutagenesis and Kinetic Studies for an Inherent Phosphatase Activity

Cited by 11 publications

References 30 publications

Structure, dynamics, and binding thermodynamics of the v‐Src SH2 domain: Implications for drug design

Structure, dynamics, and binding thermodynamics of the v‐Src SH2 domain: Implications for drug design

SH2- and SH3-mediated Interactions between Focal Adhesion Kinase and Src

Mass spectrometric approaches for the investigation of dynamic processes in condensed phase

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