analysis of jak2 catalytic function by peptide microarrays the role of the jh2 domain and v617f mutation分析jak2催化功能的多肽微阵列jh2的作用域和v617f突变.pdfVIP

Analysis of Jak2 Catalytic Function by Peptide Microarrays: The Role of the JH2 Domain and V617F Mutation 1. 2. 2 3 1 3 Arturo Sanz , Daniela Ungureanu , Tuija Pekkala , Rob Ruijtenbeek , Ivo P. Touw , Riet Hilhorst , Olli Silvennoinen2,4* 1 Department of Hematology, Erasmus MC, Rotterdam, Netherlands, 2 Institute of Biomedical Technology, University of Tampere, Tampere, Finland, 3 PamGene International BV, ’s-Hertogenbosch, The Netherlands, 4 Tampere University Hospital, Tampere, Finland Abstract Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs c