Structural Characterization of the Natively Unfolded N-Terminal Domain of Human c-Src Kinase: Insights into the Role of Phosphorylation of the Unique Domain

Institute for Research in Biomedicine, Parc Científic de Barcelona, Baldiri Reixac 10, Barcelona, Spain.
Journal of Molecular Biology (Impact Factor: 4.33). 07/2009; 391(1):136-48. DOI: 10.1016/j.jmb.2009.06.018
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The N-terminal regions of the members of Src family of non-receptor protein tyrosine kinases are intrinsically unfolded and contain the maximum sequence divergence among them. In this study, we have addressed the structural characterization by nuclear magnetic resonance of this region of 84 residues that encompasses the SH4 and the unique domains (USrc) of the human c-Src. With this aim, the backbone assignment was performed using (13)C-detected experiments that overcome the spectral resolution problems and the large number of prolines that are typical for intrinsically unfolded proteins. The analysis of the residual dipolar couplings measured for the USrc indicates the presence of a low populated helical structure in the 60-75 region. No long-range contacts between remote fragments of the chain were detected with paramagnetic relaxation enhancement experiments. The structural characterization was extended to two different phosphorylation states of USrc that encompassed three different phosphorylated sites, Ser17, Thr37, and Ser75. The structural and conformational changes upon phosphorylation were monitored through chemical shift perturbations and residual dipolar couplings, indicating that modifications occur at local level and no global rearrangements were apparent. These results suggest a scenario where phosphorylation induces a global electrostatic perturbation that could be involved in the membrane unbinding of c-Src and that could be related with the localization of the enzyme. These observations suggest the unique domain of Src kinases as a source of selectivity and reinforce the relevant role of intrinsically disordered proteins in biological processes.

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    • "The unique lipid binding region (ULBR) was discovered following NMR observations that revealed a partially structured region within the UD of Src (Pérez et al., 2009). While phosphorylation of Ser17 by PKA disturbed the interaction of the SH4 domain with lipids, phosphorylation of Thr37 and Ser75 by p25-Cdk5 decreased lipid binding by the ULBR (Pérez et al., 2009). Cross-effects were observed, suggesting a cooperative interaction of the two lipid binding regions with membranes. "
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    ABSTRACT: Members of the Src family of kinases (SFKs) are non-receptor tyrosine kinases involved in numerous signal transduction pathways. The catalytic, SH3 and SH2 domains are attached to the membrane-anchoring SH4 domain through the intrinsically disordered "Unique" domains, which exhibit strong sequence divergence among SFK members. In the last decade, structural and biochemical studies have begun to uncover the crucial role of the Unique domain in the regulation of SFK activity. This mini-review discusses what is known about the phosphorylation events taking place on the SFK Unique domains, and their biological relevance. The modulation by phosphorylation of biologically relevant inter- and intra- molecular interactions of Src, as well as the existence of complex phosphorylation/dephosphorylation patterns observed for the Unique domain of Src, reinforces the important functional role of the Unique domain in the regulation mechanisms of the Src kinases and, in a wider context, of intrinsically disordered regions in cellular processes.
    Frontiers in Genetics 06/2014; 5:181. DOI:10.3389/fgene.2014.00181
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    • "We next investigated whether the Y416 phosphorylation patterns were governed by the minimal unit of c-Src for regulating activity: the SH2, SH3, kinase, and C-tail domains [3], [31]. For this experiment, we deleted the intrinsically disordered N-terminal region (residues 1–63), which includes the unique region and a myristoylation sequence that selectively targets c-Src to the membrane, receptors and binding partners [32], [33], [34], [35]. c-Src(Δ1-63)-Emerald localized primarily in the cytosol, whereas the full length counterpart localized more extensively to the edge of the cell and internal membranous structures, consistent with its previously reported membrane/endosomal localization patterns ([36]; Fig 2A). "
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    ABSTRACT: c-Src kinase activity is regulated by phosphorylation of Y527 and Y416. Y527 phosphorylation stabilizes a closed conformation, which suppresses kinase activity towards substrates, whereas phosphorylation at Y416 promotes an elevated kinase activity by stabilizing the activation loop in a manner permissive for substrate binding. Here we investigated the correlation of Y416 phosphorylation with c-Src activity when c-Src was locked into the open and closed conformations (by mutations Y527F and Q528E, P529E, G530I respectively). Consistent with prior findings, we found Y416 to be more greatly phosphorylated when c-Src was in an open, active conformation. However, we also observed an appreciable amount of Y416 was phosphorylated when c-Src was in a closed, repressed conformation under conditions by which c-Src was unable to phosphorylate substrate STAT3. The phosphorylation of Y416 in the closed conformation arose by autophosphorylation, since abolishing kinase activity by mutating the ATP binding site (K295M) prevented phosphorylation. Basal Y416 phosphorylation correlated positively with cellular levels of c-Src suggesting autophosphorylation depended on self-association. Using sedimentation velocity analysis on cell lysate with fluorescence detection optics, we confirmed that c-Src forms monomers and dimers, with the open conformation also forming a minor population of larger mass complexes. Collectively, our studies suggest a model by which dimerization of c-Src primes c-Src via Y416 phosphorylation to enable rapid potentiation of activity when Src adopts an open conformation. Once in the open conformation, c-Src can amplify the response by recruiting and phosphorylating substrates such as STAT3 and increasing the extent of autophosphorylation.
    PLoS ONE 07/2013; 8(7):e71035. DOI:10.1371/journal.pone.0071035 · 3.23 Impact Factor
    • "The increased instrumental sensitivity leads to the development of experimental approaches to reduce the duration of NMR experiments (Schanda et al. 2006; Atreya and Szyperski 2005; Hiller et al. 2005; Jaravine et al. 2008; Malmodin and Billeter 2005; Lescop et al. 2007) and/or to exploit direct detection of nuclear spins other than 1 H (Serber et al. 2000; Bermel et al. 2003, 2006b; Shimba et al. 2005; Hu et al. 2007). Indeed, a number of exclusively heteronuclear NMR experiments based on 13 C direct detection, which take maximum advantage of the improved chemical shift dispersion of heteronuclei and of their reduced susceptibility to exchange broadening, has been developed (Bermel et al. 2006c, 2009, 2010; Bertini et al. 2011b) and proved useful in the characterization of several IDPs (Pasat et al. 2008; Csizmok et al. 2008; Pérez et al. 2009; O'Hare et al. 2009; Knoblich et al. 2009). Elegant approaches exploiting longitudinal relaxation rate enhancement (LRE) (Pervushin et al. 2002; Schanda and Brutscher 2005; Deschamps and Campbell 2006; Bermel et al. 2009b) and non-uniform sampling (NUS) (Malmodin and Billeter 2005; Hiller et al. 2005; Atreya and Szyperski 2005; Lescop et al. 2007; Jaravine et al. 2008; Kazimierczuk et al. 2010, 2012; Zawadzka-Kazimierczuk et al. 2012) of indirect evolution time space of multidimensional NMR experiments have been proposed to either reduce experimental time or improve spectral resolution; the joint use of these advancements has been reported (Bermel et al. 2009b; Bertini et al. 2011b; Novácek et al. 2011). "
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    ABSTRACT: The characterization of intrinsically disordered proteins (IDPs) by NMR spectroscopy is made difficult by the extensive spectral overlaps. To overcome the intrinsic low-resolution of the spectra the introduction of high-dimensionality experiments is essential. We present here a set of high-resolution experiments based on direct (13)C-detection which proved useful in the assignment of α-synuclein, a paradigmatic IDP. In particular, we describe the implementation of 4D HCBCACON, HCCCON, HCBCANCO, 4/5D HNCACON and HNCANCO and 3/4D HCANCACO experiments, specifically tailored for spin system identification and backbone resonances sequential assignment. The use of non-uniform-sampling in the indirect dimension and of the H-flip approach to achieve longitudinal relaxation enhancement rendered the experiments very practical.
    Journal of Biomolecular NMR 06/2012; 53(4):293-301. DOI:10.1007/s10858-012-9639-0 · 3.14 Impact Factor
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