Tyr130 phosphorylation triggers Syk release from antigen receptor by long-distance conformational uncoupling

Abstract

The Syk protein-tyrosine kinase plays a major role in signaling through the B cell receptor for antigen (BCR). Syk binds the receptor via its tandem pair of SH2 domains interacting with a doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the BCR complex. Upon phosphorylation of Tyr-130, which lies between the two SH2 domains distant to the phosphotyrosine binding sites, Syk dissociates from the receptor. To understand the structural basis for this dissociation, we investigated the structural and dynamic characteristics of the wild type tandem SH2 region (tSH2) and a variant tandem SH2 region (tSH2(pm)) with Tyr-130 substituted by Glu to permanently introduce a negative charge at this position. NMR heteronuclear relaxation experiments, residual dipolar coupling measurements and analytical ultracentrifugation revealed substantial differences in the hydrodynamic behavior of tSH2 and tSH2(pm). Although the two SH2 domains in tSH2 are tightly associated, the two domains in tSH2(pm) are partly uncoupled and tumble in solution with a faster correlation time. In addition, the equilibrium dissociation constant for the binding of tSH2(pm) to dp-ITAM (1.8 microM) is significantly higher than that for the interaction between dp-ITAM and tSH2 but is close to that for a singly tyrosine-phosphorylated peptide binding to a single SH2 domain. Experimental data and hydrodynamic calculations both suggest a loss of domain-domain contacts and change in relative orientation upon the introduction of a negative charge on residue 130. A long-distance structural mechanism by which the phosphorylation of Y130 negatively regulates the interaction of Syk with immune receptors is proposed.

Fig. 1.

The binding of Syk to a phosphorylated ITAM is inhibited by the autophosphorylation of Y130. (A) Syk-deficient DT40 cells stably expressing Myc-tagged murine Syk (WT), Syk(K396R) (KD), Syk(Y130E) or Syk(Y130F) were treated without (−) or with (+) the phosphatase inhibitor H₂O₂ (10 mM). Detergent lysates were adsorbed to an immobilized phosphopeptide corresponding to the doubly phosphorylated CD79a ITAM. Bound Syk, plotted here, was detected by Western blot analysis with an anti-Syk antibody and quantified by densitometry. (B) Myc-tagged Syk was immunoprecipitated with anti-Myc antibodies from lysates of cells preincubated in [³²P]orthophosphate and treated with H₂O₂ (Upper) or anti-IgM antibodies (Lower). The recovered protein was digested with trypsin and the resulting phosphopeptides separated electrophoretically and detected by autoradiography (Upper) or with a phosphoimager (Lower). The region of the gel containing the smaller and more rapidly migrating phosphopeptides is illustrated. The positions of the phosphotyrosines within each peptide are numbered based on the murine Syk sequence.

Fig. 2.

Spectral differences between tSH2 and tSH2_pm. (A) Overlay of part of the ¹H-¹⁵N HSQC spectra of tSH2 (red) and tSH2_pm (black) recorded at 600 MHz, 20°C. A number of peaks from residues in interdomain A and at the SH2 domain interface are observed in the tSH2_pm spectrum, but missing in the tSH2 spectrum. (B) Spectral differences are mapped onto the crystal structure for the Syk tSH2 complexed with the phosphorylated CD3ε-ITAM (PDB 1A81). Residues are colored based on HSQC peaks: observed for tSH2 but not tSH2_pm (blue), a difference in chemical shift >0.04 ppm (green), or <0.04 ppm (tan), not observed (gray). Y130 is shown in sticks. Spheres show the location of the two phosphotyrosine binding pockets. The amide group chemical shift difference between the two protein constructs, Δ_1H+15N, is calculated from the frequency difference, δ_i, of nucleus i (33): Δ_1H+15N=.

Fig. 3.

¹⁵N relaxation data (600 MHz, 293 K) for tSH2 (black) and tSH2_pm (gray). Heteronuclear NOE values (Lower) and R₂/R₁ values (Upper) are plotted on individual residues basis. The secondary structure elements in the Lower are as follows. β-strand (Black): βA (residues 15–17, 168–171), βB (residues 38–43, 191–196), βC (residues 51–57, 202–209), βDβD′ (residues 60–69, 212–221), βE (residues 74–76, 225–228), βF (residues 80–82, 232–234), βG (residues 105–107, 257–259). α-helix (Gray): αA (residues 22–31, 175–183) αB (residues 85–93,237–246). Errors were calculated by pairwise root mean square deviation of duplicate experiments.

Fig. 4.

Model of a long-distance mechanism for Y130 (red sphere) phosphorylation to down-regulate association of Syk with the antigen receptor. Phosphorylation destabilizes interdomain A structure, which alters SH2-SH2 domain orientation and retains an increased distance between pY binding sites beyond the limit permitted for bifunctional binding of ITAM.