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An Unusual Insertion in Jak2 Is Crucial for Kinase Activity and Differentially Affects Cytokine Responses

Claude Haan^1,*, Daniela C. Kroy^1,, Stefan Wüller^,, Ulrike Sommer, Tanja Nöcker, Catherine Rolvering^*, Iris Behrmann^*, Peter C. Heinrich^, and Serge Haan^2,*,

^* Life Sciences Research Unit, University of Luxembourg, Luxembourg, Luxembourg; Department of Biochemistry and Department of Pediatrics, University Hospital of Rheinish-Westphalian Technical University, Aachen, Germany; and Insitute of Biochemistry and Molecular Biology, Albert-Ludwigs University, Freiburg, Germany

The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated in myeloproliferative diseases and leukemia. Although Jaks were studied extensively for many years, the molecular mechanism of Jak activation upon cytokine stimulation of cells is still incompletely understood. In this study, we investigated the importance of an unusual insertion located within the kinase domain in Jak2. We found that the deletion of this insertion, which we named the Jak-specific insertion (JSI), totally abrogates Jak2 autophosphorylation. We further point mutated four residues within the JSI that are conserved in all Jak family members. Three of these mutants showed abrogated or reduced autophosphorylation, whereas the fourth displayed increased autophosphorylation. We found that the phosphorylation state of these mutants is not influenced by other domains of the kinase. Our data further suggest that the JSI is not required for the negative regulation of kinase activity by the suppressor of cytokine signaling proteins, SOCS. Most importantly, we show that mutations in this region differentially affect IFN- and erythropoietin signal transduction. Taken together, the dramatic effects on the phosphorylation status of Jak2 as well as the differential effects on the signaling via different cytokines highlight the importance of this unusual region for the catalytic activity of Jaks.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ C.H. and D.C.K. contributed equally to this work.

² Address correspondence and reprint requests to Dr. Serge Haan, Life Sciences Research Unit, University of Luxembourg, 162A Avenue de la Faïencerie, L-1511 Luxembourg, Luxembourg. E-mail address: serge.haan{at}uni.lu

³ Abbreviations used in this paper: FERM, 4.1, ezrin, radixin, moesin; CIS, cytokine-inducible SH2-containing protein; Epo, erythropoietin; eYFP, enhanced yellow fluorescent protein; JSI, Jak-specific insertion; FRT, Flp recombinase target; HEK, human embryonic kidney; Luc, luciferase; SOCS, suppressor of cytokine signaling; WT, wild type.

⁴ The online version of this article contains supplemental material.