Activation of the protein tyrosine kinase tyk2 by interferon alpha/beta.
We previously demonstrated that the gene tyk2 rescues the phenotype of a human mutant cell line unresponsive to alpha (IFN) and partially responsive to IFN-beta. Here, we describe functional complementation of the mutant cells with the corresponding cDNA. To characterize the putative non-receptor protein tyrosine kinase encoded by the gene tyk2 and begin to understand its functioning, we have raised polyclonal antibodies against a segment of the protein. Using these, we have identified tyk2 as a 134-kDa protein which is rapidly and transiently phosphorylated on tyrosine in response to IFN-alpha/beta and possesses an inducible kinase activity when tested in vitro. IFN-gamma has no effect on the phosphorylation state of the protein. In agreement with previous genetic evidence, these results assign a role to tyk2 in the IFN-alpha/beta signalling pathway and not in the IFN-gamma pathway. Fractionation of cell lysates have helped to localize the bulk of the protein in the cytoplasm, with a minor fraction associated with the cell membrane. Both protein pools undergo activation upon short-term IFN treatment of intact cells. Through the study of the effect of pervanadate on the phosphorylation level and the activity of tyk2, we conclude that activation of tyk2 by IFN-alpha does not require an intermediate regulatory tyrosine phosphatase.
Barbieri
G
G
Unité INSERM 276, Institut Pasteur, Paris, France.
Velazquez
L
L
Scrobogna
M
M
Fellous
M
M
Pellegrini
S
S
D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007370:Interferon Type I / Q000494:pharmacology*; D016899:Interferon-beta / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation / Q000187:drug effects; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D053634:TYK2 Kinase
D018076:DNA, Complementary; D007370:Interferon Type I; D011506:Proteins; D011994:Recombinant Proteins; D016899:Interferon-beta; D007371:Interferon-gamma; D011505:Protein-Tyrosine Kinases; D053634:TYK2 Kinase; C070139:TYK2 protein, human