Structural and Thermodynamic Characterization of the TYK2 and JAK3 Kinase Domains in Complex with CP-690550 and CMP-6

Chrencik, Jill E.; Patny, Akshay; Leung, Iris; Korniski, Brian; Emmons, Thomas L.; Hall, Troii; Weinberg, Robin A.; Gormley, Jennifer A.; Williams, Jennifer M.; Day, Jacqueline E.; Hirsch, Jeffrey L.; Kiefer, James R.; Leone, Joseph W.; Fischer, H.; Sommers, Cynthia D.; Huang, Hengming; Jacobsen, E. J.; Tenbrink, R. E.; Tomasselli, Alfredo G.; Benson, Timothy E.

doi:10.1016/j.jmb.2010.05.020

Cited by 199 publications

(190 citation statements)

References 45 publications

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“…The TYK2 kinase domain adopts the characteristic bilobate protein kinase fold and has high similarity to previously reported structures (21,25) (Fig. S3A).…”

Section: Resultssupporting

confidence: 78%

“…Although recent work has provided valuable structural information about the JAK kinase (20)(21)(22) and pseudokinase (11,13) domains, the molecular mechanism by which JAK kinase activity is regulated by the pseudokinase is poorly understood. A recent structure of the JAK1 pseudokinase (13) provides tantalizing evidence that its N terminus may act as a switch controlling JAK1 kinase activation.…”

Section: Jak1 | Jak3mentioning

confidence: 99%

See 1 more Smart Citation

Structure of the pseudokinase–kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition

Lupardus¹,

Ultsch²,

Wallweber³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

186

241

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Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state. JAK1 | JAK3H elical bundle cytokines of the interleukin and IFN families regulate a wide variety of immune and cellular growth responses (1). These signaling pathways are initiated by cytokinemediated receptor dimerization and subsequent activation of nonreceptor tyrosine kinases called Janus kinases (JAKs) that are associated with the receptor intracellular domains (2). JAK activation leads to phosphorylation of the receptor and recruitment of STAT family transcription factors, which are then phosphorylated by the JAK and translocated to the nucleus where they induce gene transcription. The JAK family consists of four members (JAK1, JAK2, JAK3, and tyrosine kinase 2 or TYK2), each of which binds a distinct set of cytokine receptor subtypes and hence has a unique gene knockout phenotype, ranging from embryonic lethality due to neuronal and erythropoietic defects (JAK1 and JAK2, respectively) (3-5), to profound immune system deficiencies (JAK3 and TYK2) (6).The JAK kinases are large (∼1,150-aa) multidomain proteins whose primary sequences were initially organized into seven JAK-homology (JH) domains that reflected distinct regions of high sequence homology (7). Subsequent structure predictions indicated that JAKs contain four distinct domains: N-terminal 4.1, Ezrin, Radixin, Moesin (FERM) and Src homology 2 (SH2) domains, followed by C-terminal pseudokinase and kinase domains (7,8) (Fig. 1A). The FERM and SH2 domains constitute the receptor-binding module (2), whereas the pseudokinase, which has a canonical kinase fold but lacks key catalytic residues, is thought to regulate kinase activity. Deletion of the pseudokinase in JAK2 and JAK3 increases basal kinase activity and deregulates signaling through cognate receptors (9, 10). Additionally, the JAK2 pseudokinase and kinase domains have been reported to coimmunoprecipitate, and coexpression of the JAK2 pseudokinase domain inhibits activity of the isolated kinase domain (10). Recent work on the JAK2 pseudokinase domain indicates that it has weak catalytic activity and autophosphorylates two inhibitory sites within the SH2-pseudokinase linker and pseudokinase...

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“…The TYK2 kinase domain adopts the characteristic bilobate protein kinase fold and has high similarity to previously reported structures (21,25) (Fig. S3A).…”

Section: Resultssupporting

confidence: 78%

Section: Jak1 | Jak3mentioning

confidence: 99%

Structure of the pseudokinase–kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition

Lupardus¹,

Ultsch²,

Wallweber³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

186

241

View full text Add to dashboard Cite

show abstract

“…However, most of the Jaks bind to their cytokine receptor through the N-terminal FERM domain, which encompasses from JH7 through a part of JH4 domain (16). In the case of Jak3, the FERM domain also interacted with the JH1 kinase domain, thereby activating it (14). Our data showed that tyrosine phosphorylation of Jak3 was necessary for its interactions with villin and that these interactions took place through direct contact between the FERM domain of Jak3 and villin (Fig.…”

Section: Discussionmentioning

confidence: 65%

“…1B, Jak3-wt autophosphorylated itself in a time-dependent manner with a t1 ⁄ 2 (the time taken to reach half of the maximum phosphorylation) of autophosphorylation at 135 s. To further confirm these, we determined the dose effect of Jak3 inhibitor CP-690550. Using crystal structure studies, it was shown that CP-690550 directly bound to the kinase domain of nonphosphorylated Jak3 (14). Fig.…”

Section: Recombinant Jak3 Autophosphorylates Itself and Transphosphormentioning

confidence: 99%

Identification of Molecular Switch Regulating Interactions of Janus Kinase 3 with Cytoskeletal Proteins

Mishra

Karanki

Kumar

2012

Journal of Biological Chemistry

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Background: Jak3 is a tyrosine kinase, and the mechanism of Jak3 interactions with cytoskeletal proteins is not known. Results: Tyrosine autophosphorylation of SH2 domain of Jak3 facilitated the interactions between the Jak3-FERM domain and cytoskeletal proteins. Conclusion:Results demonstrate the molecular mechanism of interactions between Jak3 and cytoskeletal proteins. Significance: Understanding of Jak3 functions has important implications in transplant biology, epithelial wound repair, cancer metastasis, and immune cell migration.

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“…As in other protein kinases, ATP binding may prime for critical conformational changes (40,41). Indeed, structural studies showed that the isolated JH1 domains of the four Jaks complexed with ATP-competitive inhibitors adopt an active conformation with an exposed loop (42)(43)(44). With regard to intact Jaks, specific ATP competitive inhibitors that block catalytic function also promote activation loop phosphorylation in cells (45).…”

Section: Discussionmentioning

confidence: 99%

Two Rare Disease-Associated Tyk2 Variants Are Catalytically Impaired but Signaling Competent

Gakovic

Ragimbeau

et al. 2013

The Journal of Immunology

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Tyk2 belongs to the Janus protein tyrosine kinase family and is involved in signaling of immunoregulatory cytokines (type I and III IFNs, IL-6, IL-10, and IL-12 families) via its interaction with shared receptor subunits. Depending on the receptor complex, Tyk2 is coactivated with either Jak1 or Jak2, but a detailed molecular characterization of the interplay between the two enzymes is missing. In human populations, the Tyk2 gene presents high levels of genetic diversity with >100 nonsynonymous variants being detected. In this study, we characterized two rare Tyk2 variants, I684S and P1104A, which have been associated with susceptibility to autoimmune disease. Specifically, we measured their in vitro catalytic activity and their ability to mediate Stat activation in fibroblasts and genotyped B cell lines. Both variants were found to be catalytically impaired but rescued signaling in response to IFN-α/β, IL-6, and IL-10. These data, coupled with functional study of an engineered Jak1 P1084A, support a model of nonhierarchical activation of Janus kinases in which one catalytically competent Jak is sufficient for signaling provided that its partner behaves as proper scaffold, even if inactive. Through the analysis of IFN-α and IFN-γ signaling in cells with different Jak1 P1084A levels, we also illustrate a context in which a hypomorphic Jak can hamper signaling in a cytokine-specific manner. Given the multitude of Tyk2-activating cytokines, the cell context–dependent requirement for Tyk2 and the catalytic defect of the two disease-associated variants studied in this paper, we predict that these alleles are functionally significant in complex immune disorders.

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Structural and Thermodynamic Characterization of the TYK2 and JAK3 Kinase Domains in Complex with CP-690550 and CMP-6

Cited by 199 publications

References 45 publications

Structure of the pseudokinase–kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition

Structure of the pseudokinase–kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition

Identification of Molecular Switch Regulating Interactions of Janus Kinase 3 with Cytoskeletal Proteins

Two Rare Disease-Associated Tyk2 Variants Are Catalytically Impaired but Signaling Competent

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