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 re sponsive to IFN-beta. Here, we describe functional complementation of the mutant cells with the corresponding cDNA. To characterize the puta tive non-receptor protein tyrosine kinase encoded by the gene tyk2 and begin to understand its functioning, we have raised polyclonal antibo dies against a segment of the protein. Using these, we have identified tyk2 as a 134-kDa protein which is rapidly and transiently phosphoryl ated on tyrosine in response to IFN-alpha/beta and possesses an induci ble kinase activity when tested in vitro. IFN-gamma has no effect on t he phosphorylation state of the protein. In agreement with previous ge netic evidence, these results assign a role to tyk2 in the IFN-alpha/b eta signalling pathway and not in the IFN-gamma pathway. Fractionation of cell lysates have helped to localize the bulk of the protein in th e cytoplasm, with a minor fraction associated with the cell membrane. Both protein pools undergo activation upon shortterm IFN treatment of intact cells. Through the study of the effect of pervanadate on the ph osphorylation level and the activity of tyk2, we conclude that activat ion of tyk2 by IFN-alpha does not require an intermediate regulatory t yrosine phosphatase.