A dual role for the kinase-like domain of the tyrosine kinase Tyk2 in interferon-α signaling

A dual role for the kinase-like domain of the tyrosine kinase Tyk2 in interferon-α signaling

^*Laboratoire de Signalisation des Cytokines, Institut Pasteur, Paris 75724 Cedex 15, France; and ^†Centre National de la Recherche Scientifique EP2030, Institut de Génétique Moléculaire de Montpellier, 34293 Montpellier Cedex 5, France

Edited by George R. Stark, Cleveland Clinic Foundation, Cleveland, OH, and approved May 26, 2000 (received for review March 23, 2000)

Abstract

Tyrosine kinases of the Janus kinase family initiate cellular responses through their association with receptors for α-helical cytokines. In addition to a tyrosine kinase domain, these enzymes possess a kinase-like (KL) domain, whose function remains elusive. To investigate the role of the KL domain of Tyk2 in interferon-α/β signaling, we transfected a library of Tyk2 cDNAs containing random point mutations in KL into Tyk2-negative cells and selected for loss-of-function Tyk2 mutants. Four such mutants, V584D, G596V, H669P, and R856G, were identified through this screen. Like the wild-type Tyk2, the mutant proteins were able to sustain the level of IFNAR1 receptor protein. However, all four mutants were incapable of restoring high-affinity interferon-α binding in Tyk2-negative cells and were also catalytically impaired, even when transiently overexpressed. Interferon-α induced phosphorylation, and gene expression could be detected in V584D- or G596V-expressing cells, but not in H669P- or R856G-expressing cells. Furthermore, H669P and R856G proteins were constitutively highly phosphorylated. All together, our findings demonstrate that an intact KL domain is essential for the intrinsic catalytic activity of Tyk2 and for the establishment of a high-affinity interferon-α receptor complex.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: Laboratoire de Signalisation des Cytokines, Institut Pasteur, 25 rue du Docteur Roux, Paris 75724 Cedex 15, France. E-mail: pellegri{at}pasteur.fr.
This paper was submitted directly (Track II) to the PNAS office.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.160130297.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.160130297
Abbreviations:

JAK,

Janus kinase;

STAT,

signal transducers and activators of transcription;

KL,

kinase-like;

TK,

tyrosine kinase;

6TG,

6-thioguanine;

WT,

wild type;

IU,

international units;

IRK,

insulin receptor kinase