Journal of Molecular Biology
Regular articleStructural basis for syk tyrosine kinase ubiquity in signal transduction pathways revealed by the crystal structure of its regulatory SH2 domains bound to a dually phosphorylated ITAM peptide1
Introduction
ZAP-70 and Syk comprise a family of hematopoietic cell specific protein tyrosine kinases (PTKs) that are required for antigen and antibody receptor function (reviewed by Chan and Shaw 1995, Wange and Samelson 1996, Reth and Wienands 1997, Kurosaki 1997). While ZAP-70 is expressed in T and natural killer cells, Syk is expressed in B cells, mast cells, polymorphonuclear leukocytes, platelets, macrophages, and immature T cells. The absence of either ZAP-70 or Syk results in arrested T and B cell development and in functional defects of a variety of immune receptors including the T cell antigen receptor (TCR), the B cell antigen receptor (BCR), and the receptors for IgG and IgE (FcγRI and FcϵRI; Costello et al 1996, Zhang et al 1996, Crowley et al 1997; and reviewed by Cheng & Chan, 1997 and by Kurosaki, 1997). In addition, this family of PTKs has been implicated in activating NK cells Brumbaugh et al 1997, Lanier et al 1998 and signaling by non-immune receptors such as G-protein coupled and integrin receptors Wan et al 1996, Gao et al 1997, Poole et al 1997. The signaling components of the antigen and Ig receptors contain sequence motifs known as ITAMs (for Immunoreceptor Tyrosine-based Activation Motifs) which have the consensus sequence YxxL/I-x7/8-YxxL/I (Reth, 1989). Phosphorylation of both tyrosine residues within the ITAM by the Src-family PTKs is required for efficient interaction of ZAP-70 and Syk with the receptor subunits and for receptor function Iwashima et al 1994, Isakov et al 1995, Bu et al 1995, Chen et al 1996, Kurosaki et al 1995.
The Syk family of PTKs is characterized by a domain structure consisting of two N-terminal Src-homology 2 (SH2) domains and a C-terminal kinase domain separated from the SH2 domains by a linker or hinge region (reviewed by Chan & Shaw, 1995). The localization of ZAP-70 and Syk to the receptor is mediated through the high affinity interaction between the SH2 domains of ZAP-70 and Syk with the ITAM phosphorylated on both tyrosine residues Isakov et al 1995, Bu et al 1995, Chen et al 1996. The inability of either tyrosine residue within the ITAM to be phosphorylated or mutation of either SH2 domain within ZAP-70 or Syk decreases the avidity of this interaction by >100-fold and results in a non-functional antigen receptor Kong et al 1995, Kurosaki et al 1995, Isakov et al 1995, Bu et al 1995. The co-localization of ZAP-70 and Syk to the receptor complex, in part, permits the membrane-localized Src-PTKs to contribute to the phosphorylation and enzymatic activation of ZAP-70 and Syk Chan et al 1995, Kurosaki et al 1995, El-Hillal et al 1997.
While ZAP-70 and Syk have similar structures and play overlapping roles in T cell biology, these two PTKs also exhibit many different characteristics. First, the Syk kinase domain is ∼100-fold more active than ZAP-70 (Latour et al., 1996). Second, the enzymatic activation of ZAP-70 is primarily dependent upon the trans-phosphorylation by Src-PTKs (reviewed by Chan & Shaw, 1995). In contrast, the enzymatic activity of Syk can be augmented both by Src-PTKs and by the binding of dually phosphorylated ITAM peptides Rivera and Brugge 1995, Rowley et al 1995, Kurosaki et al 1995, El-Hillal et al 1997. Third, expression of Syk is more ubiquitous amongst hematopoietic cells and Syk has been shown to be activated by not only a large variety of immune response receptors, but also by cytokines, integrins, thrombin and G protein-coupled receptors Minami et al 1995, Corey et al 1994, Wan et al 1996, Taniguchi et al 1993, Clark et al 1994. Fourth, recent evidence has indicated that activation of Syk by non-immune response receptors such as integrin and G protein-coupled receptors may require only one functional SH2 domain for activity and therefore may not involve a classical ITAM/Syk interaction Gao et al 1997, Wan et al 1996.
Although the distinct patterns of expression of ZAP-70 and Syk may explain their selective involvement in signal transduction pathways, differences in ITAM or phosphotyrosine-recognition and binding specificity by the SH2 domains of these proteins may also account for their differences in function. Recently, the X-ray crystal structure of the liganded tandem SH2 domain of ZAP-70 was determined, providing the structural basis for the obligatory requirement of ZAP-70 for dually phosphorylated ITAMs and insights into ITAM recognition by this protein (Hatada et al., 1995). We present here the structure of the tandem SH2 domain of Syk bound to a dually phosphorylated ITAM. In contrast to ZAP-70, where one of the two phosphotyrosine binding pockets is shared by both SH2 domains, this binding pocket in Syk is self-contained within the N-terminal SH2 domain, suggesting that the N and C-terminal SH2 domains can function independently. In addition, the two SH2 domains of Syk display a remarkable flexibility in their relative orientation, suggesting that Syk may accommodate a greater variety of spacing sequences between the ITAM phosphotyrosines as well as singly phosphorylated non-classical ITAM ligands. Hence, this structure provides a structural basis for the broader spectrum of recognition processes mediated by the Syk-PTK and addresses the issue of specificity in the recruitment of Syk to immune and non-immune receptors.
Section snippets
Structure determination
The crystal structure of the Syk-tandem-SH2 domain complexed with a dually phosphorylated ITAM peptide, derived from the CD3ϵ chain of the T cell receptor, was determined by multiple isomorphous replacement and anomalous scattering to a resolution of 3.0 Å (Figure 1 and Materials and Methods). The asymmetric unit contains six copies of the bound tandem-SH2 domain (Figure 2), which were built independently (details in Materials and Methods). Therefore, this structure reports six independent
Conclusions
From the crystal structures of the SH2 domains of the Syk and ZAP-70 kinases complexed with ITAM peptides, common structural themes emerge which can be seen as the general features of the mechanism of ITAM recognition and binding. Firstly, the tandem SH2 domain of the Syk family of proteins bind tyrosyl-phosphorylated ITAM sequences with a defined polarity determined by the relative positioning of the two SH2 domains. Hence, the N-terminal pYxxL/I motif of the ITAM binds to the C-terminal SH2
Purification, crystallization and data collection
The tandem SH2 domain fragment of Syk (residues 7 to 269) was expressed and purified as described by Bu et al. (1995). The pure protein was complexed with the CD3ϵ-chain ITAM peptide (sequence PDpYEPIRKGQRDLpYSGLNQR, from Quality Controlled Biochemicals) at a ratio of 1:1.1 protein to ligand, and concentrated to typically 35 mg/ml. Crystals were grown at room temperature using the hanging drop vapor diffusion method (Mc Pherson, 1990). Drops of 2 μl protein were mixed with 2 μl of 9 to 12%
Acknowledgements
We thank Ian Wilson and Jack Johnson (Scripps Research Institute) for advice on NCS averaging, C.S. Ricard for advice on protein purification, S. Korolev and A.B. Herr for help in data collection, M. Hatada (ARIAD Pharmaceuticals) for providing the ZAP-70 tandem SH2 domain coordinates, the staff of Beamline 7.1 at SSRL for assistance during data collection, and F.S. Mathews for comments on the manuscript. This work was supported by Pfizer Inc. (G.W.), by funds from the Washington University
References (55)
- et al.
Phosphoinositide 3-kinase and p72syk noncovalently associate with the low affinity Fcγ receptor on human platelets through an immunoreceptor tyrosine-based activation motif
J. Biol. Chem.
(1996) - et al.
Interaction of phosphorylated FcϵRIγ immunoglobulin receptor tyrosine activation motif-based peptides with dual and single SH2 domains of p72syk. Assessment of binding parameters and real time binding kinetics
J. Biol. Chem.
(1996) - et al.
Regulation of the protein tyrosine kinase pp72syk by platelet agonists and the integrin αIIbβ3
J. Biol. Chem.
(1994) - et al.
Crystal structure of the tyrosine phosphatase SHP-2
Cell
(1998) - et al.
Reconstitution of Syk function by the ZAP-70 protein tyrosine kinase
Immunity
(1995) - et al.
Structures of the SH2 and SH3 domains
Curr. Opin. Struct. Biol.
(1993) Molecular mechanisms in B cell antigen receptor signaling
Curr. Opin. Immunol.
(1997)- et al.
Differential intrinsic enzymatic activity of Syk and Zap-70 protein-tyrosine kinases
J. Biol. Chem.
(1996) - et al.
Protein tyrosine kinase Syk is associated with and activated by the IL-2 receptorpossible link with the c-myc induction pathway
Immunity
(1995) - et al.
Conformations of polypeptides and proteins
Advan. Protein Chem.
(1968)
Protein tyrosine kinase p72syk is activated by thrombin and is negatively regulated through Ca2+ mobilization in platelets
J. Biol. Chem.
Binding of a high affinity phosphotyrosyl peptide to the src SH2 domaincrystal structures of the complexed and peptide-free forms
Cell
Complex complexessignaling at the TCR
Immunity
Functional role for Syk tyrosine kinase in natural killer cell-mediated natural cytotoxicity
J. Exp. Med.
X-PLOR (Version 3. 1) Manual
The free R valuea novel statistical quantity for assessing the accuracy of crystal structures
Nature
Slow-cooling protocols for crystallographic refinement by simulated annealing
Acta Crystallog. sect. A
Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance
Proc. Natl Acad. Sci. USA
The CCP4 suite of programs for protein crystallography
Acta Crystallog. sect. D
Regulation of antigen receptor signal transduction by protein tyrosine kinases
Curr. Opin. Immunol.
Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function
EMBO J.
PTKs in thymocyte development
Curr. Opin. Immunol.
Granulocyte colony-stimulating factor receptor signaling involves the formation of a three-component complex with Lyn and Syk protein-tyrosine kinases
Proc. Natl Acad. Sci. USA
Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cell
Oncogene
A critical role for Syk in signal transduction and phagocytosis mediated by Fcγ receptors on macrophages
J. Exp. Med.
Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwave length anomalous diffraction methods
Methods Enzymol.
Recognition of a high affinity phosphotyrosyl peptide by the Src homology 2 domain of p56lck
Nature
Cited by (176)
Syk regulates the haemocyte autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 in oyster antibacterial immunity
2023, Fish and Shellfish Immunology ReportsSpatial requirements for ITAM signaling in an intracellular natural killer cell model membrane
2022, Biochimica et Biophysica Acta - General SubjectsAn evolutionary divergent thermodynamic brake in ZAP-70 fine-tunes the kinetic proofreading in T cells
2022, Journal of Biological ChemistryCitation Excerpt :The corresponding kinetic behavior also showed a single exponential decay as observed in our experiment (Fig. 3H). Syk is less selective than ZAP-70 and activated by a wide range of ITAM sequences in cells of innate and adaptive immune systems (10, 17, 20, 36, 46–48). Sequence analysis shows that most PBP and allosteric network residues in the regulatory module are conserved between Syk and ZAP-70 (Fig. 5, A and B).
Back on the scene: Advances and challenges in CD3-related drugs in tumor therapy
2022, Drug Discovery Today
- 1
Edited by D. Rees