The Atlantic salmon protein tyrosine kinase Tyk2: Molecular cloning, modulation of expression and function Mehrdad Sobhkhez, Tom Hansen, Dimitar B. Iliev, Astrid Skjesol, Jorunn B. Jørgensen ⇑ Norwegian College of Fisheries Science, University of Tromsø, N-9037 Tromsø, Norway article info Article history: Received 10 May 2013 Revised 9 July 2013 Accepted 11 July 2013 Available online 18 July 2013 Keywords: JAK STAT Interferons Innate immunity Tyrosine kinase 2 (Tyk2) abstract Tyk2, a member of the Janus Kinase (JAK) family of protein tyrosine kinases, is required for interferon-a/b binding and signaling in higher vertebrates. Currently, little is known about the role of the different JAKs in signaling responses to interferon (IFN) in lower vertebrates including fish. In this paper we report the identification and characterization of Atlantic salmon (Salmo salar) Tyk2. Four cDNA sequences, two containing an open reading frame encoding full-length Tyk protein and two with an up-stream in frame stop codon, were identified. The deduced amino acid sequences of the salmon full-length Tyk2 proteins showed highest identity with Tyk2 from other species and their transcripts were ubiquitously expressed. Like in mammals the presented data suggests that salmon Tyk2 is auto- phosporylated when ectopically expressed in cells. In our experiments, full-length salmon Tyk2 overex- pressed in CHSE-cells phosphorylated itself, while both a kinase-deficient mutant and the truncated Tyk2 (Tyk-short) were inactive. Interestingly, the overexpression of full length Tyk2 was shown to up-regulate the transcript levels of the IFN induced gene Mx, thus indicating the involvement of salmon Tyk2 in the salmon IFN I pathway. Ó 2013 Published by Elsevier Ltd. 1. Introduction Cytokines are secretory proteins playing essential roles in in- nate and adaptive immunity. Some of them, including interleukins, interferons (IFNs) and hematopoietic growth factors activate the Janus kinase (JAK)–signal transducers and activators of the tran- scription (STAT) pathway (Gadina et al., 2001). In this pathway, cytokine binding results in receptor oligomerization, which initi- ates the activation of JAK kinases (Lemmon and Schlessinger, 1994). The activated JAKs phosphorylate the receptor cytoplasmic domains, which creates docking sites for SH2-containing signaling proteins, including STATs (Rawlings et al., 2004). The JAKs form one subgroup of non-receptor protein tyrosine kinases and are found in mammals, birds, insects (Yamaoka et al., 2004) and also in some fish species (Conway et al., 1997; Guo et al., 2009; Leu et al., 2000; Ma et al., 2007). In mammals there are four JAK-family- members identified (JAK1–JAK3 and Tyk2), three of which; JAK1, JAK2 and Tyk2, are expressed in almost all tissues, while the expression of JAK3 is limited to lymphoid tissues (Yamaoka et al., 2004). Their amino acid sequence shows seven conserved JAK homology domains (JH1–7) specific for this group of kinases (see Fig. 1), and is unique in having two tandemly arranged kinase do- mains: a carboxyterminal tyrosine kinase domain (JH1), followed by JH2, referred to as a pseudo-kinase domain. JH2 has sequence similarity with other tyrosine kinases but it has no catalytic activ- ity. A study using Tyk2 loss-of-function mutants could demon- strate that JH2 are essential for Tyk2 catalytic activity and for restoring high affinity interactions between IFNa and its receptor (Yeh et al., 2000). The amino terminal region (JH3–7) includes the FERM (four point one, ezrin, radixin, moesin) domain (JH5 and JH6), which directs stable association with membrane proxi- mal receptor motifs. Additionally, the amino terminal part harbors the Src Homology 2 domain (SH2). In general the N-terminal re- gion of Tyk2 plays an important role in the IFNAR1 stability (Gauz- zi et al., 1997). JAKs are activated when interaction between cytokines and cytokine receptors brings the receptor subunits in proximity of each other, and this leads to phosphorylation of tyro- sine residues on the JAKs (auto- and trans-phosphorylation) as well as on intracellular domains of the receptors. The latter constitutes docking site for signal molecules and especially STAT molecules (Aaronson and Horvath, 2002; Brierley and Fish, 2005; Platanias, 2005). Tyk2 was the first JAK-family member shown to be essential for cytokine signaling. In the seminal studies a mutant cell line 0145-305X/$ - see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.dci.2013.07.008 Abbrevations: aa, amino acid; bp, base pair; JAK, Janus kinase; Tyk2, Tyrosine kinase 2; IFN, Interferon; ss, Salmo salar; STAT, signal transducers and activators of the transcription; JH, janus homology; HK, head kidney; ISG’s, interferon responsive genes; ISRE, interferon stimulated response element. ⇑ Corresponding author. Tel.: +47 95095754. E-mail address: jorunn.jorgensen@uit.no (J.B. Jørgensen). Developmental and Comparative Immunology 41 (2013) 553–563 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci