We analysed the interaction of gp130, the common signal-transducing receptor chain of interleukin (IL)-6 type cytokines, with Jak1, the Janus family kinase which is crucial for signal transduction of this group of cytokines. With a truncated chimaeric IL-5Rβ–gp130 receptor expressed in COS-7 cells, we show that the membrane-proximal 69 amino acids are sufficient to mediate Jak1 binding and activation. Deletion of box2 drastically reduced binding of endogenous, but not of overexpressed, Jak1. Several point mutations in the membrane-proximal region of gp130 (W652A, P671/P672A, F676A, Y683F, where W, A, P, F and Y are tryptophan, alanine, proline, phenylalanine and tyrosine) did not affect Jak1 association. However, stimulation of chimaeric receptors with the mutations P671/P672A and F676A in the interbox1/box2 region resulted in a reduced activation of STAT (signal transducer and activator of transcription) transcription factors. Most importantly, signalling by the receptor with the box1 mutation W652A was totally abrogated. Although this mutation did not affect Jak1 association, stimulation-dependent phosphorylation of Jak1 was prevented. The W652 mutation acts dominantly, since no signalling occured even when only a single cytoplasmic chain of a gp130 dimer contained the mutation. Our data demonstrate that the mere proximity of Jaks in an activated receptor complex is not sufficient to mediate their activation. Rather, it seems that parts of the receptor, including the box1 region, are involved in positioning Jaks correctly so that ligand-induced receptor dimerization and reorientation can lead to their mutual activation and subsequently to downstream signalling events.
- box1/box2 region
- Jak association
- tyrosine kinase
Abbreviations used: EMSA, electrophoretic-mobility-shift assay; EPOR, erythropoietin receptor; Jak, Janus kinase; LIF, leukaemia inhibitory factor; OSM (R), oncostatin M (receptor); sIL-6R, soluble IL-6 receptor; STAT, signal transducer and activator of transcription; W652A etc., Trp652 → Ala mutant etc.; C/EBP, CCAAT-enhancer-binding protein; ECL® (Amersham), enhanced chemiluminescence; FCS, fetal-calf serum; SIE, c-sis-inducible element.
- The Biochemical Society, London ©2002