The amino-terminal region of Tyk2 sustains the level of interferon α receptor 1, a component of the interferon α/β receptor

Abstract

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-␣ (IFN-␣) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-␣. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-␣ receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-␣. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.The intracellular protein-tyrosine kinases of the Janus kinase (JAK) family play an essential role in cytokine signaling: they interact with receptor components and undergo tyrosine phosphorylation and enzymatic activation upon ligand binding. Their activation is the first step of a cascade of intracellular phosphorylation events ultimately leading to the transcriptional activation of target genes (1, 2). Signaling through the receptor for type I interferons (IFN-␣ and -␤) requires two members of the JAK family, Tyk2 and JAK1 (3, 4). Both enzymes are associated with the receptor, which is composed of IFN-␣ receptor (IFNAR) 1 (5) and IFNAR2-2, the longer splice variant of the IFNAR2 gene (6, 7). Tyk2 was shown to interact with the membrane-proximal region of IFNAR1 (8, 9), and JAK1 with IFNAR2-2 (10, 11). The stoichiometry of the activated receptor͞JAK complex is not known and might differ for the different type I IFNs. Ligand-induced dimerization or oligomerization of the receptor components leads to asymmetric trans-phosphorylation and consequent catalytic activation of Tyk2 and JAK1 (4,12,13).Tyk2 shares with the other members of the JAK family a unique structural framework (see Fig. 1A) comprising seven conserved JAK homology (JH) regions (14). The most Cterminal one (JH1) is a tyrosine kinase (TK) domain, which is flanked by the JH2 or kinase-like (KL) domain of unknown function. The remaining five blocks of homology (JH3 to JH7) extend toward the N terminus of the protein and exhibit variable degrees of conservation among the family members, the most conserved being JH4 with a central core of 18 identical...

Two Distinct Domains Within the N-Terminal Region of Janus Kinase 1 Interact with Cytokine Receptors

“…Therefore, it is critical to define the domains in Jaks and cytokine receptors responsible for this interaction to mimic or abrogate the activation of cytokine systems with specific drugs. Some reports have indicated that the N-terminal region, containing the JH7-6 domains, of Jak2, Jak3, and Tyk2 is critical for the association with receptors (42)(43)(44)(45)(46)(47)(48)(49). Our data (Fig.…”

“…The JH7-6 domains of Tyk2 interact with IFN-␣R. Although a direct interaction between the JH5-4-3 domains of Tyk2 and IFN-␣R␣ has not been established, these domains are also required for kinase activation by the receptor (47)(48)(49).…”

The human papilloma virus (HPV)-18 E6 oncoprotein physically associates with Tyk2 and impairs Jak-STAT activation by interferon-α

“…Second, the JH 6 -JH 7 domains of Tyk2 are also required for the interaction with the cytoplasmic portion of the IFNAR1 subunit ( Figure 8B). Interestingly, the JH 6 -JH 7 domains are required for e cient phosphorylation of IFNAR1 in vitro and for rendering the kinase activable by IFN-a (Gauzzi et al, 1997). Third, tyrosine phosphorylation of Tyk2 is diminished in cells expressing HPV-18 E6 after stimulation with IFN-a ( Figure 5A).…”

“…To this end, we used wt Tyk2 and E6 proteins bearing the vesicular stomatitis virus glycoprotein (VSV-G) (Gauzzi et al, 1997) and FLAG epitope in their C-terminus end respectively. VSV-GTyk2 and FLAG-E6 proteins were co-transfected in HeLa cells using the vaccinia virus/T7 system and their expression levels were detected by immunoblotting with either anti-VSV-G (Figure 7, top row) or anti-FLAG antibodies (second from the top row).…”

IL-3 signaling and the role of Src kinases, JAKs and STATs: a covert liaison unveiled

“…Considering the variety of interactions and functions performed by the JAK kinase family members it seemed plausible that these domains may be responsible for some key interactions with other proteins, which could act as their substrates. Recent studies indicate that the amino terminal regions are also involved in receptor binding (Gauzzi et al, 1997;Chen et al, 1997).…”