Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 pseudokinase domain negatively regulates the activity of the JH1 kinase domain. The structures of wild-type JH2 and of the dominant V617F mutant provide a solid foundation for discerning the mechanism of action of the JH2 domain in normal JAK signaling and in dysregulated signaling that causes disease. The protein tyrosine kinase JAK2 mediates signaling through numerous cytokine receptors. JAK2 possesses a pseudokinase domain (JH2) and a tyrosine kinase domain (JH1). Through unknown mechanisms, JH2 regulates the catalytic activity of JH1, and hyperactivating mutations in the JH2 region of human JAK2 cause myeloproliferative neoplasms (MPNs). We showed previously that JAK2 JH2 is, in fact, catalytically active. Here we present crystal structures of human JAK2 JH2, including both wild type and the most prevalent MPN mutant, V617F. The structures reveal that JH2 adopts the fold of a prototypical protein kinase but binds Mg-ATP noncanonically. The structural and biochemical data indicate that the V617F mutation rigidifies α-helix C in the N lobe of JH2, facilitating trans-phosphorylation of JH1. The crystal structures of JH2 afford new opportunities for the design of novel JAK2 therapeutics targeting MPNs.

Signal transducer and activator of transcription 1 (STAT1) is a critical mediator of interferon-γ (IFN-γ)–induced transcription that is regulated through posttranslational modifications and through transacting proteins such as protein inhibitor of activated STAT1 (PIAS1). PIAS proteins have been shown to function as E3-type small ubiquitin-like modifier (SUMO) ligases, and sumoylation has been identified as a modulatory mechanism for several transcription factors. Here we show that STAT1 is subject to SUMO-1 modification, and sumoylation occurs in vivo and in vitro at a single, evolutionary conserved amino acid residue Lys703. Members of the PIAS family of proteins were found to strongly stimulate sumoylation of STAT1. Furthermore, activation of STAT1 by IFN-γ or pervanadate induced SUMO-1 conjugation. Mutation of Lys703 in STAT1 resulted in increased IFN-γ–mediated transactivation, suggesting a negative regulatory function for sumoylation. These results indicate that STAT1 is covalently modified by SUMO-1 in cytokine signaling and that PIAS proteins promote SUMO-1 conjugation to STAT1.