Mutations in the EGFR kinase are a cause of non-small-cell lung cancer. To understand their mechanism of activation and effects on drug binding, we studied the kinetics of the L858R and G719S mutants and determined their crystal structures with inhibitors including gefitinib, AEE788, and a staurosporine. We find that the mutations activate the kinase by disrupting autoinhibitory interactions, and that they accelerate catalysis as much as 50-fold in vitro. Structures of inhibitors in complex with both wild-type and mutant kinases reveal similar binding modes for gefitinib and AEE788, but a marked rotation of the staurosporine in the G719S mutant. Strikingly, direct binding measurements show that gefitinib binds 20-fold more tightly to the L858R mutant than to the wild-type enzyme.
Appropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM domain directly binds the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation.
Summary Upon stimulation by pathogen-associated inflammatory signals, the atypical IκB kinase TBK1 induces type-I interferon expression and modulates NF-κB signaling. Here we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a novel dimeric assembly, created by an extensive network of interactions among the kinase, ubiquitin-like (ULD) and scaffold/dimerization (SDD) domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on Lysine 30 and Lysine 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog IKKε, but not in the canonical IκB kinase IKKβ, which assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IκB kinases in normal and disease physiology and will further development of more specific inhibitors that may be useful as anti-cancer or anti-inflammatory agents.
Jak (Janus kinase) family nonreceptor tyrosine kinases are central mediators of cytokine signaling. The Jak kinases exhibit distinct cytokine receptor association profiles and so transduce different signals. Jak3 expression is limited to the immune system, where it plays a key role in signal transduction from cytokine receptors containing the common gammachain, ␥c. Patients unable to signal via ␥c present with severe combined immunodeficiency (SCID). The finding that Jak3 mutations result in SCID has made it a target for development of lymphocytespecific immunosuppressants. Here, we present the crystal structure of the Jak3 kinase domain in complex with staurosporine analog AFN941. The kinase domain is in the active conformation, with both activation loop tyrosine residues phosphorylated. The phosphate group on pTyr981 in the activation loop is in part coordinated by an arginine residue in the regulatory C-helix, suggesting a direct mechanism by which the active position of the C-helix is induced by phosphorylation of the activation loop. Such a direct coupling has not been previously observed in tyrosine kinases and may be unique to Jak kinases. The crystal structure provides a detailed view of the Jak3 active site and will facilitate computational and structure-directed approaches to development of Jak3-specific inhibitors. ( IntroductionThe Janus kinase (Jak) family of cytoplasmic tyrosine kinases are essential for signal transduction from a wide variety of cell-surface receptors. There are 4 members of the family in vertebrates: Jak1, Jak2, Jak3, and tyrosine kinase 2 (Tyk2). 1,2 Jak kinases share a characteristic domain architecture, which includes an aminoterminal FERM domain (Band 4.1, Ezrin, Radixin, Moesin homology domain), an src homology 2 (SH2)-like region, a pseudokinase domain, and a carboxy-terminal kinase domain. Parts of these structural domains have historically been termed Jak homology (JH) domains 1 through 7, based on primary sequence alignments. The FERM domain mediates association with the cytoplasmic region of cytokine receptors and may also participate in catalytic regulation. The function and activity of the SH2-like region is unclear. The pseudokinase (or JH2) domain is unique to Jak kinases. This domain is thought to have a protein kinase fold but to lack catalytic activity, as residues critical for phosphotransfer are absent. The pseudokinase domain has been shown to be intrinsic to the autoregulation of Jak kinases via a direct interaction with the kinase domain. 3 The JH1 kinase domain lies at the C-terminus and is a functional tyrosine kinase. To date, no 3-dimensional structure has been reported for any portion of any of the Jak kinases.A wide variety of cytokine receptor superfamily members signal via the Jak/Stat (signal transducer and activator of transcription) pathway, including granulocyte colony-stimulating factor (G-CSF), thrombopoietin, the interferons, erythropoietin, and the interleukins. The Jak/Stat pathway is consequently involved in regulation of diverse cell proc...
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