A JAK1 JAK2 CHIMERA CAN SUSTAIN ALPHA-INTERFERON AND GAMMA-INTERFERONRESPONSES/

Cell lines that are mutated in interferon (IPN) responses have been cr itical in establishing an essential role for the JAK family of nonrece ptor tyrosine kinases in interferon signalling. Mutant gamma 1A cells have previously been shown to be complemented by overexpression of JAK 2. Here, it is shown that these cells carry a defect in, and can also be complemented by, the beta-subunit of the IFN-gamma receptor, consis tent with the hypothesis that the mutation in these cells affects JAK2 -receptor association. In contrast, mutant gamma 2A cells lack detecta ble JAK2 mRNA and protein, By using gamma 2A cells, the role of variou s domains and conserved tyrosine residues of JAK2 in IFN-gamma signall ing was examined. Individual mutation of six conserved tyrosine residu es, mutation of a potential phosphatase binding site, or mutation of t he arginine residue in the proposed SH2-like domain had no apparent ef fect on signalling in response to IFN-gamma. Results with deletion mut ants, however, indicated that association of JAK2,vith the IFN-gamma R 2 subunit requires the amino-terminal region but not the pseudokinase domain, Consistent with this, in chimeras with JAK1, the JAK2 amino-te rminal region was required for receptor association and STAT1 activati on. Conversely, a JAK1-JAK2 chimera with the amino-terminal domains of JAK1 linked to the pseudokinase and kinase domains of JAK2 is capable of reconstituting JAK-STAT signalling in response to IFN-alpha and -g amma in mutant U4C cells lacking JAK1. The specificity of the JAKs may therefore lie mainly in their structural interaction with different r eceptor and signalling proteins rather than in the substrate specifici ty of their kinase domains.