A Novel Mode of Gleevec Binding Is Revealed by the Structure of Spleen Tyrosine Kinase

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine → phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine. Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine → phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine. Syk family members include two human proteins, Syk (spleen tyrosine kinase) and its closest relative Zap-70 (70-kDa ζ chain-associated protein). Syk and Zap-70 are non-receptor tyrosine kinases characterized by two N-terminal SH2 adapter domains, a linker region, and a C-terminal catalytic domain. Syk and Zap-70 share similar functions in the transduction of immunoreceptor signals. Antigen binding to immunoreceptors leads to phosphorylation of two tyrosine side chains in the cytoplasmic immunoreceptor tyrosine-based activation motif. Phosphorylation of the immunoreceptor tyrosine-based activation motif recruits either Syk or Zap-70 via their SH2 domains (for a review see Ref. 1Latour S. Veillette A. Curr. Opin. Immunol. 2001; 13: 299-306Crossref PubMed Scopus (171) Syk of the in the in in Immunol. PubMed Scopus and PubMed Scopus Zap-70 a similar in signaling A. Immunol. PubMed Scopus Syk is in a of its is in hematopoietic cells. have that Syk is for Immunol. 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Antigen binding to immunoreceptors leads to phosphorylation of two tyrosine side chains in the cytoplasmic immunoreceptor tyrosine-based activation motif. Phosphorylation of the immunoreceptor tyrosine-based activation motif recruits either Syk or Zap-70 via their SH2 domains (for a review see Ref. 1Latour S. Veillette A. Curr. Opin. Immunol. 2001; 13: 299-306Crossref PubMed Scopus (171) Syk of the in the in in Immunol. PubMed Scopus and PubMed Scopus Zap-70 a similar in signaling A. Immunol. PubMed Scopus Syk is in a of its is in hematopoietic cells. have that Syk is for Immunol. 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PubMed Scopus of mutants are to of the activation loop and the active conformation seen in the structure of Gleevec to the of Abl PubMed Scopus however, the weaker Gleevec binding mode in the existence of a of mutants that the conformation not the conformation. is that a of the mutants to activation loop and are by of Gleevec in the is with for the observed of or not Gleevec binds to phosphorylated Abl or the of Abl the cis-conformation is not and have to The however, for The structure of the Syk kinase domain why Syk catalytic does not require activation loop Syk S. Veillette A. PubMed Scopus or Syk the activation loop catalytic S. PubMed Scopus Immunol. PubMed Scopus PubMed Scopus The structure of the isolated Zap-70 kinase catalytic domain suggests that adopts an active conformation S. 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