Pérez Y et al. (2013), Lipid binding by the Unique and SH3 domains of ...

XB-ART-46699

Sci Rep 2013 Jan 01;3:1295. doi: 10.1038/srep01295.

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Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism.

Pérez Y , Maffei M , Igea A , Amata I , Gairí M , Nebreda AR , Bernadó P , Pons M .

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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.

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Species referenced: Xenopus
Genes referenced: fbxw4 mapk1 pc src

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	Figure 1. Lipid binding by the Unique domain of c-Src (a) Overlay of 1H-15N HSQC NMR spectra USrc alone (red) and in the presence of DMPC/DHPC bicelles (blue), and DMPG/DHPC bicelles (grey).(b) Combined 1H-15N lipid-induced chemical shift changes per residue. The color code is the same as in (a). Total lipid concentration was 8% w/v and the ratio of long chain lipids to DHPC (q) was 0.8. (c) Binding of USrc to immobilized lipids, detected by immunoblotting with anti-Strep-tag HRP (TG triglyceride, DAC diacylglyceride, PA phosphatidic acid, PS phosphatidylserine, PE phosphatidylethanolamine, PC phosphatidylcholine, PG phosphatidylglycerol, CL cardiolipin, PtdIns phosphatidylinositol). (d) Schematic representation of the structure and charge of the lipid bicelles used.
	Figure 2. Lipid binding by Unique-SH3 (USH3) and isolated SH3 domains.Lipid binding by the USH3 construct (a, b) and the isolated SH3 domain (c,d,e). (a, d)) Combined 1H-15N chemical shift perturbations induced by the presence of 8% w/v of DMPG/DHPC bicelles (q = 0.8). (b,c) Immunoblots showing the lipid binding specificity (see Fig. 1c for the identity of the lipids and to compare with the isolated Unique domain). (e) Residues perturbed in presence of lipids are highlighted in yellow on the SH3 surface (PDB code: 1SHG). The polyproline binding site is located in the opposite side and indicated by the presence of a polyproline ligand.
	Figure 3. Interaction between Unique and SH3 domains (a,b) Combined absolute values 1H-15N NMR chemical shift differences between linked and isolated domains.(a) USH3 versus USrc. (b) USH3 versus SH3. (c) Intensity ratios of NH cross-peaks from MTLS-labeled (A59C/USH3 at pH 7.0) between paramagnetic and diamagnetic (DTT reduced) forms. (d) SH3 residues perturbed by interaction with the Unique domain are highlighted in orange. The polyproline binding site is located in the opposite site and indicated by the presence of a polyproline ligand. (e) Cartoon representation of the interaction between SH3 and Unique domains observed by PRE.
	Figure 4. Allosteric effect of polyproline peptide binding on the Unique-SH3 interaction.(aâe) Expansion of HSQC spectra showing residues A55 and E150 (acting as control) at molar rations of the Unique-SH3 construct (USH3) to Ac-VSLARRPLPPLP-OH of (a) 0, (b) 0.2, (c) 0.4, (d) 0.7, and (e) 1.0. (f) Expansion of HSQC spectrum of the isolated Unique domain (USrc) containing residue A55. All spectra were recorded at 25Â°C and 800âMHz. (g) Cartoon representation depicting the fact that he fast equilibrium between the open-close interaction between the Unique and SH3 domains is cancelled by the interaction of a polyproline (PP) peptide with the SH3 domain in spite of the fact that the two binding sites are separated.
	Figure 5. Effect of phosphorylation on lipid binding by USrc.Combined 1H-15N NMR shifts induced by DMPG/DHPC bicelles (8% w/v total lipid concentration, q = 0.8) in USrc unphosphorylated (a,b, gray), monophosphorylated at S17 (a, blue), and diphosphorylated at T37 and S75 (b, blue).
	Figure 6. Calmodulin binding by the Unique domain of c-Src.Two different expansions of 1H-15N HSQC NMR spectra obtained during the titration of USrc with Ca2+-calmodulin (CaM) are shown in panels a and b. (c) Combined absolute values of 1H-15N chemical shift changes induced by 2-fold excess of Ca2+-calmodulin in USrc residues.
	Figure 7. Sequence alignment of the Unique domain of Src.Sequence alignment of the Unique domains of human, mouse, chicken and Xenopus laevis Src is shown.Green boxes highlight residues S17, T37 and S75 that are known to be phosphorylated in vitro. Orange box shows the position of the 6 aminoacids of the ULBR that were mutated in the oocytes maturation experiment (see Fig. 8).
	Figure 8. Effect of Src mutants on Xenopus oocyte maturation.The effect of the injection of oocytes with mRNAs encoding wild-type c-Src (green), AAA mutant (blue), EAE mutant (magenta), or water (orange) is compared with that of non-injected oocytes (yellow).(a) Percentage of oocytes that underwent maturation, as determined germinal vesicle breakdown (GVBD), at different times after the addition of progesterone. The inset shows the percentages of morphologically normal oocytes at the end of the experiment, when 100% of the control oocytes treated with progesterone reached GVBD. (b) Oocyte appearance 2 and 4âh after GVBD. About half of the matured oocytes that express AAA or EAE mutants show progressive depigmentation starting 2âh after GVBD. (c) Analysis of the expression levels of wild-type and mutants forms of human c-Src and endogenous Xenopus c-Src. Oocyte lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-human c-Src (top panel), anti-Xenopus c-Src (middle panel) and anti-MPK1 (bottom panel) as a loading control.

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