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Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.

ABSTRACT: Mutations in FAM20A cause tooth enamel defects known as Amelogenesis Imperfecta (AI) and renal calcification. We previously showed that Fam20A is a secretory pathway pseudokinase and allosterically activates the physiological casein kinase Fam20C to phosphorylate secreted proteins important for biomineralization (Cui et al., 2015). Here we report the nucleotide-free and ATP-bound structures of Fam20A. Fam20A exhibits a distinct disulfide bond pattern mediated by a unique insertion region. Loss of this insertion due to abnormal mRNA splicing interferes with the structure and function of Fam20A, resulting in AI. Fam20A binds ATP in the absence of divalent cations, and strikingly, ATP is bound in an inverted orientation compared to other kinases. Fam20A forms a dimer in the crystal, and residues in the dimer interface are critical for Fam20C activation. Together, these results provide structural insights into the function of Fam20A and shed light on the mechanism by which Fam20A mutations cause disease.

SUBMITTER: Cui J

PROVIDER: S-EPMC5413348 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.

Cui Jixin J Zhu Qinyu Q Zhang Hui H Cianfrocco Michael A MA Leschziner Andres E AE Dixon Jack E JE Xiao Junyu J

eLife 20170422

Mutations in <i>FAM20A</i> cause tooth enamel defects known as Amelogenesis Imperfecta (AI) and renal calcification. We previously showed that Fam20A is a secretory pathway pseudokinase and allosterically activates the physiological casein kinase Fam20C to phosphorylate secreted proteins important for biomineralization (Cui et al., 2015). Here we report the nucleotide-free and ATP-bound structures of Fam20A. Fam20A exhibits a distinct disulfide bond pattern mediated by a unique insertion region. ...[more]

PMID: 28432788

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Project description:Janus kinase 3 (JAK3) tyrosine kinase has a central role in the control of lymphopoiesis, and mutations in JAK3 can lead to either severe combined immunodeficiency or leukemia and lymphomas. JAK3 associates with the common gamma chain (?c) receptor and functions in a heteromeric signaling pair with JAK1. In IL-2 signaling JAK1 is the effector kinase for STAT5 phosphorylation but the precise molecular regulatory mechanisms of JAK1 and JAK3 and their individual domains are not known. The pseudokinase domain (JAK homology 2, JH2) of JAK3 is of particular interest as approximately half of clinical JAK3 mutations cluster into it. In this study, we investigated the role of JH2s of JAK1 and JAK3 in IL-2R signaling and show that STAT5 activation requires both JH1 and JH2 of JAK1, while both JH1 and JH2 in JAK3 are specifically required for the cytokine-induction of cellular signaling. Characterization of recombinant JAK3 JH2 in thermal shift assay shows an unstable protein domain, which is strongly stabilized by ATP binding. Unexpectedly, nucleotide binding to JAK3 JH2 was found to be cation-independent. JAK3 JH2 showed higher nucleotide binding affinity in MANT-ATP and fluorescent polarization competition assays compared to the other JAK JH2s. Analysis of the functional role of ATP binding in JAK3 JH2 in cells and in zebrafish showed that disruption of ATP binding suppresses ligand-independent activation of clinical JAK3 gain-of-function mutations residing in either JH2 or JH1 but does not inhibit constitutive activation of oncogenic JAK1. ATP-binding site mutations in JAK3 JH2 do not, however, abrogate normal IL-2 signaling making them distinct from JH2 deletion or kinase-deficient JAK3. These findings underline the importance of JAK3 JH2 for cellular signaling in both ligand-dependent and in gain-of-function mutation-induced activation. Furthermore, they identify the JH2 ATP-binding site as a key regulatory region for oncogenic JAK3 signaling, and thus a potential target for therapeutic modulation.

Dataset Information

Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.

Publications

Structure of Fam20A reveals a pseudokinase featuring a unique disulfide pattern and inverted ATP-binding.

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