JAK1 N-terminus binds to conserved Box 1 and Box 2 motifs of cytokine receptor common beta subunit but signal activation requires JAK1 C-terminus.
The human interleukin-3 receptor (hIL-3R) consists of a unique alpha subunit (hIL-3Ralpha) and a common beta subunit (betac). Binding of IL-3 to IL-3R activates Janus kinases JAK1 and JAK2. Our previously study showed that JAK2 and JAK1 were constitutively associated with the hIL-3Ralpha and betac subunits, respectively. In this study, we further demonstrate that JAK2 binds to the intracellular domain of hIL-3Ralpha and JAK1 binds to the Box 1 and Box 2 motifs of betac using GST-hIL-3R fusion proteins in pull-down assays. JAK1 mutational analysis revealed that its JH7-3 domains bound directly to the Box 1 and Box 2 motifs of betac. We further examined the role of JAK1 JH7-3 domains in JAK1 and JAK2-mediated signaling using the CDJAKs fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1), JAK2 (CDJAK2), or JAK1-JH7-3 domains (CDJAK1-JH7-3) as intracellular domains. Anti-CD16 antibody crosslinking of wild type fusion proteins CDJAK1 with CDJAK2 could mimic IL-3 signaling, however, the crosslinking of fusion proteins CDJAK1-JH7-3 with CDJAK2 failed to activate downstream proteins. These results suggest that the JAK1-JH7-3 domains are required for betac interaction and abolish wild type JAK1 and JAK2-mediated signaling.
Huang
Huei-Mei
HM
Graduate Institute of Cell and Molecular Biology, Taipei Medical University, Taipei, Taiwan. cmbhhm@tmu.edu.tw
Lee
Ya-Li
YL
Chang
Ting-Wei
TW
D020816:Amino Acid Motifs; D000818:Animals; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D053648:Cytokine Receptor Common beta Subunit / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D053650:Interleukin-3 Receptor alpha Subunit / Q000378:metabolism; D053613:Janus Kinase 1 / Q000737:chemistry* / Q000378:metabolism*; D053614:Janus Kinase 2 / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D003432:Cross-Linking Reagents; D053648:Cytokine Receptor Common beta Subunit; D053650:Interleukin-3 Receptor alpha Subunit; D053613:Janus Kinase 1; D053614:Janus Kinase 2