Activating mutations in the cytokine receptor/JAK2 signaling axis are found at high frequency in myeloproliferative disorders (MPD). Lnk, an SH2-containing adaptor protein, is a negative regulator of several hematopoietic cytokine receptors including MPL and EpoR. Here, we assessed whether Lnk can attenuate the activity of mutant MPLW515L, JAK2V617F and JAK2K539L found in MPD patients. Lnk overexpression in Ba/F3-MPLW515L cells inhibited cytokine-independent growth, while suppression of Lnk in UT7-MPLW515L cells enhanced proliferation. Lnk-mediated growth inhibition was associated with downregulation of JAK/STAT, MAPK and PI3K signaling pathways. Similarly, Lnk inhibited cytokine-independent growth conferred by JAK2V617F and JAK2K539L in Ba/F3-EpoR cells. Following thrombopoietin stimulation, Lnk became tyrosyl-phosphorylated and associated with activated wild-type (WT) MPL and MPLW515L at the plasma membrane of Ba/F3 cells. An SH2 mutant Lnk (R392E) failed to bind and inhibit WT MPL and MPLW515L, demonstrating that the SH2 domain is essential for Lnk down-modulation of the receptors. The Lnk-MPL interaction was also detected with endogenously expressed proteins from cultured bone marrow cells. A series of C-terminally truncated Lnk constructs were used to determine which Lnk regions are required for Lnk inhibition of JAK2. Experiments in 293T cells indicated that Lnk SH2 domain binds to phosphorylated JAK2. In addition, other Lnk regions associated with non-phosphorylated JAK2, and these interactions were critical for Lnk inhibition of JAK2V617F and JAK2K539L constitutive activation. Our data suggest a model wherein Lnk downregulation of the receptor/JAK2 signaling involves two mechanisms; one is inhibition of the cytokine receptor utilizing JAK2, the second is direct suppression of JAK2 kinase activity. Furthermore, while the receptor mediated inhibition requires the SH2 domain, direct inhibition of JAK2 mutants, V617F and K539L, relies on other Lnk domains. Further elucidating the molecular mechanisms underlying Lnk inhibition of signaling pathways abnormally activated by oncogenic alleles, will provide insight into the pathogenesis of MPD and may have therapeutic value.

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