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. 2009 Mar 13;284(11):6773-81.
doi: 10.1074/jbc.M807531200. Epub 2009 Jan 12.

Acute lymphoblastic leukemia-associated JAK1 mutants activate the Janus kinase/STAT pathway via interleukin-9 receptor alpha homodimers

Tekla Hornakova  1 , Judith Staerk, Yohan Royer, Elisabetta Flex, Marco Tartaglia, Stefan N Constantinescu, Laurent Knoops, Jean-Christophe Renauld
Affiliations

Acute lymphoblastic leukemia-associated JAK1 mutants activate the Janus kinase/STAT pathway via interleukin-9 receptor alpha homodimers

Tekla Hornakova et al. J Biol Chem. .

Abstract

Activating mutations in JAK1 have been reported in acute lymphoblastic leukemias, but little is known about the mechanisms involved in their constitutive activation. Here, we studied the ability of JAK1 V658F and A634D to activate the Janus kinase (JAK)/STAT pathway upon ectopic expression in HEK293 cells alone or together with the other components of the interleukin-9 receptor complex (IL-9Ralpha, gammac, and JAK3). Expression of JAK1 mutants alone failed to trigger STAT activation, but co-expression of the IL-9Ralpha chain promoted JAK1 mutant phosphorylation and STAT activation. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Ralpha involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with a functional IL-9Ralpha to activate STAT factors. Several lines of evidence indicated that IL-9Ralpha homodimerization was involved in this process. IL-9Ralpha variants with mutations of the JAK-interacting BOX1 region not only failed to promote JAK1 activation but also acted as dominant negative forms reverting the effect of wild-type IL-9Ralpha. Coimmunoprecipitation experiments also showed the formation of IL-9Ralpha homodimers. Interestingly, STAT activation was partially inhibited by expression of gammac, suggesting that overlapping residues are involved in IL-9Ralpha homodimerization and IL-9Ralpha/gammac heterodimerization. Co-expression of wild-type JAK3 partially reverted the inhibition by gammac, indicating that JAK3 cooperates with JAK1 mutants within the IL-9 receptor complex. Similar results were observed with IL-2Rbeta. Taken together, our results show that IL-9Ralpha and IL-2Rbeta homodimers efficiently mediate constitutive activation of ALL-associated JAK1 mutants.

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Figures

FIGURE 1.
FIGURE 1.
ALL-associated JAK1 mutants (V658F and A634D) induce constitutive STAT3 activation in the presence of IL-9Rα. HEK293 cells were transiently transfected with different JAK1 constructs alone or in combination with IL-9Rα or the mutated IL-9Rα Y116F. The STAT3-responsive pGL3-pap1 construct was used as luciferase reporter. 4 h post-transfection, cells were incubated for 20 h with or without IL-9 before the luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments.
FIGURE 2.
FIGURE 2.
Y107A mutation in the FERM domain of JAK1 mutants abolishes IL-9Rα-mediated constitutive STAT3 activation. COS-7 (A) or HEK293 (B) cells were transiently co-transfected with empty vector or different JAK1 constructs with intact or mutated Y107A FERM domain (V658F/Y107A or A634D/Y107A) and with or without IL-9Rα. The STAT3-responsive pGL3-pap1 construct was used as luciferase reporter. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments in COS-7 and HEK293 cells.
FIGURE 3.
FIGURE 3.
Co-expression of γc inhibits IL-9Rα-mediated JAK1 phosphorylation and STAT3 activation. A, HEK293 cells were transiently co-transfected with empty vector, JAK1 wild-type or V658F, γc, or/and IL-9Rα in addition to the STAT3-responsive luciferase reporter pGL3-pap1. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments. B, HEK293 cells were transiently co-transfected with different empty vector, JAK1 constructs, γc, or/and IL-9Rα. 24 h post-transfection, 106 cells were lysed and subjected to Western blot analysis. Phosphorylation of JAK1 and STAT3 was detected using specific anti-pJAK1 Tyr-1022/1023 and anti-pSTAT3 Tyr-705 antibodies. Membranes were reprobed with anti-JAK1, anti-STAT3 and anti-β-actin antibodies as control. Similar results were obtained in two independent experiments. C, HEK293 cells were transiently co-transfected with empty vector, JAK1 wild-type or V658F, and IL-9Rα with or without γc in addition to the STAT3-responsive luciferase reporter pGL3-pap1. One day post-transfection, an aliquot of cells was used to assess cell surface expression of IL-9Rα by FACS analysis using anti-human IL-9Rα antibody followed with phycoerythrin-conjugated streptavidin (SAPE).
FIGURE 4.
FIGURE 4.
Co-expression of JAK3 partially abrogates the γc inhibitory effect on IL-9Rα-mediated STAT3 activation by JAK1 V658F. HEK293 cells were transiently co-transfected with JAK1 wild-type or V658F, γc, IL-9Rα, and/or with JAK3 as the last component of IL-9R complex in addition to the STAT3-responsive luciferase reporter pGL3-pap1. 4 h post-transfection cells were incubated for 20 h with or without IL-9 and subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments.
FIGURE 5.
FIGURE 5.
Dominant negative effect of IL-9Rα BOX1 mutant on constitutive JAK1 V658F phosphorylation and STAT3 activation mediated by IL-9Rα WT. A, HEK293 cells were transiently co-transfected with JAK1 wild-type or V658F and different IL-9Rα constructs in addition to the STAT3-responsive luciferase reporter pGL3-pap1. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments. B, HEK293 cells were transiently co-transfected with JAK1 wild-type or V658F and different IL-9Rα constructs. 24 h post-transfection 106 cells were lysed and subjected to Western blot analysis. Phosphorylation of JAK1 and STAT3 was detected using specific anti-pJAK1 Tyr-1022/1023 and anti-pSTAT3 Tyr-705 antibodies. Membranes were reprobed with anti-JAK1, anti-STAT3, and anti-β-actin antibodies as control. Similar results were obtained in three independent experiments.
FIGURE 6.
FIGURE 6.
IL-9Rα BOX1 mutant, but not TPOR or EPOR, inhibits IL-9Rα-mediated activation of STAT3 by JAK1 V658F. HEK293 cells were transiently co-transfected with JAK1 wild-type or V658F, IL-9Rα wild-type, and/or BOX1 mutant or/and with the thrombopoietin or erythropoietin receptors in addition to the STAT3-responsive luciferase reporter. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in two independent experiments.
FIGURE 7.
FIGURE 7.
Co-immunoprecipitation shows evidence of IL-9Rα homodimerization. HEK293 cells were transiently co-transfected with Myc- and/or HA-tagged IL-9Rα or HA-tagged EPOR. 24 h post-transfection, cellular extracts were immunoprecipitated (IP) with an anti-Myc antibody and analyzed by Western blot with anti-HA antibodies to detect co-immunoprecipitation of IL-9Rα. Anti-Myc antibodies were used as the control, and total lysates were analyzed with the same antibodies. Similar results were obtained in three independent experiments.
FIGURE 8.
FIGURE 8.
Both IL-9Rα and IL-2Rß allows for JAK1 V658F- and JAK1 A634D-induced constitutive activation of STAT5. A, HEK293 cells were transiently co-transfected with empty vector, JAK1 wild-type, V658F or A634D, IL-9Rα and/or γc in addition to the STAT5-responsive luciferase reporter pLHRE. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments. B, the same experiment was performed with IL-2Rβ. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments.
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