Phosphorylation of unique domains of Src family of kinases

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

Structure of the K-turn U4 RNA: a combined NMR and SANS study

K-turn motifs are universal RNA structural elements providing a binding platform for proteins in several cellular contexts. Their characteristic is a sharp kink in the phosphate backbone that puts the two helical stems of the protein-bound RNA at an angle of 60°. However, to date no high-resolution structure of a naked K-turn motif is available. Here, we present the first structural investigation at atomic resolution of an unbound K-turn RNA (the spliceosomal U4-Kt RNA) by a combination of NMR and small-angle neutron scattering data. With this study, we wish to address the question whether the K-turn structural motif assumes the sharply kinked conformation in the absence of protein binders and divalent cations. Previous studies have addressed this question by fluorescence resonance energy transfer, biochemical assays and molecular dynamics simulations, suggesting that the K-turn RNAs exist in equilibrium between a kinked conformation, which is competent for protein binding, and a more extended conformation, with the population distribution depending on the concentration of divalent cations. Our data shows that the U4-Kt RNA predominantly assumes the more extended conformation in the absence of proteins and divalent cations. The internal loop region is well structured but adopts a different conformation from the one observed in complex with proteins. Our data suggests that the K-turn consensus sequence does not per se code for the kinked conformation; instead the sharp backbone kink requires to be stabilized by protein binders

Hubble Space Telescope Combined Strong and Weak Lensing Analysis of the CLASH Sample: Mass and Magnification Models and Systematic Uncertainties

We present results from a comprehensive lensing analysis in HST data, of the complete CLASH cluster sample. We identify new multiple-images previously undiscovered allowing improved or first constraints on the cluster inner mass distributions and profiles. We combine these strong-lensing constraints with weak-lensing shape measurements within the HST FOV to jointly constrain the mass distributions. The analysis is performed in two different common parameterizations (one adopts light-traces-mass for both galaxies and dark matter while the other adopts an analytical, elliptical NFW form for the dark matter), to provide a better assessment of the underlying systematics - which is most important for deep, cluster-lensing surveys, especially when studying magnified high-redshift objects. We find that the typical (median), relative systematic differences throughout the central FOV are $\sim40\%$ in the (dimensionless) mass density, $\kappa$, and $\sim20\%$ in the magnification, $\mu$. We show maps of these differences for each cluster, as well as the mass distributions, critical curves, and 2D integrated mass profiles. For the Einstein radii ($z_{s}=2$) we find that all typically agree within $10\%$ between the two models, and Einstein masses agree, typically, within $\sim15\%$. At larger radii, the total projected, 2D integrated mass profiles of the two models, within r\sim2\arcmin, differ by $\sim30\%$. Stacking the surface-density profiles of the sample from the two methods together, we obtain an average slope of $d\log (\Sigma)/d\log(r)\sim-0.64\pm0.1$, in the radial range [5,350] kpc. Lastly, we also characterize the behavior of the average magnification, surface density, and shear differences between the two models, as a function of both the radius from the center, and the best-fit values of these quantities.Comment: 35 pages (20 main text pages, plus 15 pages for additional figures and tables); 2 Tables, 17 Figures. V3: accepted version; some minor corrections and additions made. V4: corrected several entries in Table 2. All mass models and magnification maps are made publicly available for the communit

The SH3 domain acts as a scaffold for the N-terminal intrinsically disordered regions of c-Src

Regulation of c-Src activity by the intrinsically disordered Unique domain has been recently demonstrated. However, its connection with the classical regulatory mechanisms is still missing. Here we show that the Unique domain is part of a long loop closed by the interaction of the SH4 and SH3 domains. The conformational freedom of the Unique domain is further restricted through direct contacts with SH3 that are allosterically modulated by binding of a poly-proline ligand in the presence and in the absence of lipids. Our results highlight the scaffolding role of the SH3 domain for the c-Src N-terminal intrinsically disordered regions and suggest a connection between the regulatory mechanisms involving the SH3 and Unique domains

Multi-phosphorylation of the intrinsically disordered unique domain of c-Src studied by in-cell and real-time NMR

Intrinsically disordered regions (IDRs) are preferred sites for post-translational modifications essential for regulating protein function. The enhanced local mobility of IDRs facilitates their observation by NMR spectroscopy in vivo. Phosphorylation events can occur at multiple sites and respond dynamically to changes in kinase-phosphatase networks. Here we used real-time NMR spectroscopy to study the effect of kinases and phosphatases present in Xenopus oocytes and egg extracts on the phosphorylation state of the 'unique domain' of c-Src. We followed the phosphorylation of S17 in oocytes, and of S17, S69, and S75 in egg extracts by NMR spectroscopy, MS, and western blotting. Addition of specific kinase inhibitors showed that S75 and S69 are phosphorylated by CDKs (cyclin-dependent kinases) differently from Cdk1. Moreover, although PKA (cAMP-dependent protein kinase) can phosphorylate S17 in vitro, this was not the major S17 kinase in egg extracts. Changes in PKA activity affected the phosphorylation levels of CDK-dependent sites, thus suggesting indirect effects of kinase-phosphatase networks. This study provides a proof-of-concept of the use of real-time in vivo NMR spectroscopy to characterize kinase/phosphatase effects on intrinsically disordered regulatory domains

CLASH: Mass Distribution in and around MACS J1206.2-0847 from a Full Cluster Lensing Analysis

We derive an accurate mass distribution of the galaxy cluster MACS J1206.2-0847 (z=0.439) from a combined weak-lensing distortion, magnification, and strong-lensing analysis of wide-field Subaru BVRIz' imaging and our recent 16-band Hubble Space Telescope observations taken as part of the Cluster Lensing And Supernova survey with Hubble (CLASH) program. We find good agreement in the regions of overlap between several weak and strong lensing mass reconstructions using a wide variety of modeling methods, ensuring consistency. The Subaru data reveal the presence of a surrounding large scale structure with the major axis running approximately north-west south-east (NW-SE), aligned with the cluster and its brightest galaxy shapes, showing elongation with a \sim 2:1 axis ratio in the plane of the sky. Our full-lensing mass profile exhibits a shallow profile slope dln\Sigma/dlnR\sim -1 at cluster outskirts (R>1Mpc/h), whereas the mass distribution excluding the NW-SE excess regions steepens further out, well described by the Navarro-Frenk-White form. Assuming a spherical halo, we obtain a virial mass M_{vir}=(1.1\pm 0.2\pm 0.1)\times 10^{15} M_{sun}/h and a halo concentration c_{vir} = 6.9\pm 1.0\pm 1.2 (\sim 5.7 when the central 50kpc/h is excluded), which falls in the range 4 <7 of average c(M,z) predictions for relaxed clusters from recent Lambda cold dark matter simulations. Our full lensing results are found to be in agreement with X-ray mass measurements where the data overlap, and when combined with Chandra gas mass measurements, yield a cumulative gas mass fraction of 13.7^{+4.5}_{-3.0}% at 0.7Mpc/h (\approx 1.7r_{2500}), a typical value observed for high mass clusters.Comment: Accepted by ApJ (30 pages, 17 figures), one new figure (Figure 10) added, minor text changes; a version with high resolution figures available at http://www.asiaa.sinica.edu.tw/~keiichi/upfiles/MACS1206/ms_highreso.pd

An oxygen-sensitive toxin-antitoxin system

The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin-antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SOxH- containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle

Synthesis and biological evaluation of N-cyanoalkyl-, Naminoalkyl-, and N-guanidinoalkyl-substituted 4-aminoquinoline derivatives as potent, selective, brain permeable antitrypanosomal agents

YesCurrent drugs against human African trypanosomiasis (HAT) suffer from several serious drawbacks. The search for novel, effective, brain permeable, safe, and inexpensive antitrypanosomal compounds is therefore an urgent need. We have recently reported that the 4-aminoquinoline derivative huprine Y, developed in our group as an anticholinesterasic agent, exhibits a submicromolar potency against Trypanosoma brucei and that its homo- and hetero-dimerization can result in to up to three-fold increased potency and selectivity. As an alternative strategy towards more potent smaller molecule anti-HAT agents, we have explored the introduction of ω-cyanoalkyl, ω-aminoalkyl, or ω-guanidinoalkyl chains at the primary amino group of huprine or the simplified 4-aminoquinoline analogue tacrine. Here, we describe the evaluation of a small in-house library and a second generation of newly synthesized derivatives, which has led to the identification of 13 side chain modified 4-aminoquinoline derivatives with submicromolar potencies against T. brucei. Among these compounds, the guanidinononyltacrine analogue 15e exhibits a 5-fold increased antitrypanosomal potency, 10-fold increased selectivity, and 100-fold decreased anticholinesterasic activity relative to the parent huprine Y. Its biological profile, lower molecular weight relative to dimeric compounds, reduced lipophilicity, and ease of synthesis, make it an interesting anti-HAT lead, amenable to further optimization to eliminate its remaining anticholinesterasic activity.Wellcome Trus

Phosphorylation of unique domains of Src family of kinases

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

Lipid binding by the unique and SH3 domains of c-Src suggests a new regulation mechanism

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase,SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation