Direct binding to and tyrosine phosphorylation of the alpha subunit of the type I interferon receptor by p135tyk2 tyrosine kinase.

Abstract

Binding of type I interferons (IFNs) to their receptors induces rapid tyrosine phosphorylation of multiple proteins, including the a and jB subunits of the receptor, the polypeptides that form the transcriptional activator ISGF3a (Statll3, Stat84, and Stat9l), and the pl35'yk2 and Jak-1 tyrosine kinases. In this report, we demonstrate that the a subunit of the type I IFN receptor (IFN-R) corresponds to the product of a previously cloned receptor subunit cDNA and, further, that the pl35cyk2 tyrosine kinase directly binds and tyrosine phosphorylates this receptor subunit. Glutathione S-transferase (GST) fusion proteins encoding the different regions of the cytoplasmic domain of the a subunit can bind the pl35'yk2 contained in human cell lysates. The association between the a subunit and Tyk2 was demonstrated by immunoblotting with anti-Tyk2 and antiphosphotyrosine antibodies and by using an in vitro kinase assay. Analogous experiments were then performed with recombinant baculoviruses encoding constitutively active Jak family tyrosine kinases. In this case, pl35tk2, but not Jak-1 or Jak-2 protein, binds to the GST-IFN-R proteins, suggesting that the interaction between these two proteins is both direct and specific. We also demonstrate that Tyk2, from extracts of either IFNa-treated human cells or insect cells infected with the recombinant baculoviruses, can catalyze in vitro phosphorylation of GST-IFN-R protein in a specific manner. Deletion mutants of the GST-IFN-R protein were used to localize both the binding and tyrosine phosphorylation site(s) to a 46-amino-acid juxtamembrane region of the a subunit, which shows sequence homology to functionally similar regions of other cytokine receptor proteins. These data support the hypothesis that the Tyk2 protein functions as part of a receptor complex to initiate intracellular signaling in response to type I IFNs.

Induction of early response genes by interferons, interleukins, and growth factors by the tyrosine phosphorylation of latent transcription factors

“…One component of the IFNa receptor was cloned several years ago, but it clearly did not bind IFNP or most types of IFNa (61). Recently, it has been shown that this chain is a component of the IFNa receptor, can directly interact with the tyk2 kinase (62), and is tyrosine phosphorylated in cells treated with IFNa (63,64). Another subunit of the I F N d P receptor has been shown to bind both IFNa and IFNP, and it constitutively associates with Jakl (65).…”

Homodimerization and intermolecular tyrosine phosphorylation of the Tyk‐2 tyrosine kinase

“…The antiphosphotyrosine and anti-JAK-I antibodies were obtained from UBI (Lake Placid, NY) and the anti-tyk2 monoclonal antibody was purchased from Transduction Laboratories. The rabbit sera anti-Tyk2 have been previously described [8].…”

“…Type I IFNs (IFN~/fl/oJ) bind to a cell surface receptor (IFN~R or type I IFN-R) composed of two chains: the ~ and fl subunits [1][2][3]. The ~ subunit corresponds to the cDNA termed IFNAR cloned by Uz6 et al [4,5]. The IFNc~flR cDNA recently cloned by Novick et al [6] corresponds to a short form of the fl subunit (termed fls), while a long form (termed ilL) has been recently cloned in our laboratory and is reported elsewhere [7].…”

“…The IFNc~flR cDNA recently cloned by Novick et al [6] corresponds to a short form of the fl subunit (termed fls), while a long form (termed ilL) has been recently cloned in our laboratory and is reported elsewhere [7]. The cytoplasmic domain of the ~ subunit directly associates with Tyk-2 [5,8]. Similarly, Jak-I appears to coprecipitate with the 1~ subunit [6], although a direct interaction has not been demonstrated.…”

“…Similarly, Jak-I appears to coprecipitate with the 1~ subunit [6], although a direct interaction has not been demonstrated. Type I IFN binding to the receptor induces rapid tyrosine phosphorylation of the Tyk-2 [5,9] and Jak-1 kinases [9-11], the components of the transcription factor ISGF3t (Statle, Statlfl, and Stat2) [12][13][14][15][16] (reviewed in [17]), and the ~ and ]3 subunits of the IFNczR [18][19][20], (E.N. Constantinescu et al, Proc.…”