Adaptor Protein LNK Binds to and Is Phosphorylated by JAK3 and May Serve as a Scaffold for JAK3 Autophosphorylation In the Absence of An Appropriate Cytokine Receptor (2010)

Abstract Abstract 2785 The adaptor protein LNK has an important role during the development and maturation of cells in the hematopoietic system. LNK KO mice have an expansion of hematopoietic stem cells (HSCS) and an increase in circulating neutrophils, platelets, as well as immature B cells. On the other hand, overexpression of WT LNK under the control of a lymphocyte-specific expression vector results in an impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. Recently, LNK PH domain mutations were described in myeloproliferative neoplasms (MPN). The non-receptor tyrosine kinase JAK3 is expressed mainly in hematopoietic cells and together with JAK1, transmits signals from cytokine receptors of the IL-2 family. While JAK3 loss of function causes severe combined immune deficiency (SCID), JAK3 activating mutations were described in AML, particularly in acute megakaryocytic leukemia (AMKL). The importance of both LNK and JAK3 in lymphopoiesis, coupled with the finding that LNK binds to another JAK family member (JAK2), has led us to study whether LNK and JAK3 interact. LNK and JAK3 co- immunoprecipitated (co-IP) in the following leukemic cell lines- HEL, NALM6 (containing both LNK and WT JAK3), CMK (containing both LNK and an activating JAK3 mutant) and not in K562 (no JAK3). WT-JAK3 and WT LNK also co-IP when overexpressed in 293T cells, and this binding was associated with phosphorylation of LNK. To determine which domain of LNK is responsible for JAK3 binding, WT JAK3 was overexpressed in 293T cells with the following LNK mutants: 1. point mutation in the SH2 domain (R392E); 2. Deletion of the SH2 domain (del SH2); 3. Deletion of the PH and SH2 (del SH2/PH) domains; 4. Point mutation in the PH domain, recently found in a patient with a JAK2 V617F negative MPN (E208Q); 5. LNK SNP (rs3184504) described in GWAS to associate with an increased risk of autoimmune disease and which also causes an amino acid change in the LNK PH domain (W262R). LNK binding was retained in the E208Q and W262R mutants, markedly reduced in the R392E and del SH2 mutants and completely abolished with the del SH2/PH mutant, suggesting that LNK SH2 domain is important for the binding of LNK and WT-JAK3, although some binding may occur even in its absence. The effect of the LNK mutants R392E, E208Q and W262R on WT-JAK3 and LNK phosphorylation was also studied in 293T cells. Notably, WT-JAK3 was phosphorylated in the presence of WT-LNK and the E208Q and W262R mutants and not in the presence of the R392E mutant or an empty vector; also, the WT-LNK, LNK E208Q and W262R were phophorylated and the LNK R392E was not. These results suggest that WT-JAK3 phosphorylates LNK and that the LNK SH2 domain is important for this to occur. Interestingly, we found that the LNK PH domain mutant (E208Q) was much more phosphorylated than the WT- LNK when co-expressed with WT-JAK3. A similar result was obtained when LNK E208Q was co-expressed in 293T cells with vectors expressing an activating JAK3 mutant- A572V and the JAK2 V617F mutant but not when it was co-expressed with a BCR-ABL vector. This, again, supports the fact that direct binding underlines LNK phosphorylation by JAK3, as was previously shown for JAK2, while the BCR-ABL kinase does not bind LNK. The LNK PH domain mutation E208Q may cause a conformational change in LNK, allowing better access to tyrosine residues, or a change in LNK localization. The finding in 293T cells that WT-JAK3 is phophorylated only in the presence of LNK that retains a PH domain is intriguing. The other LNK family members- SH2B1 and APS were previously shown to homo- and heterodimerize. LNK contains a dimerization domain and we speculate that LNK homodimerization may serve as a scaffold for WT-JAK3 in the absence of a cytokine receptor, enabling JAK3 autophosphorylation. Since LNK PH domain mutations were recently described in MPN and may involve activation of the JAK2-STAT pathway we hypothesized that similar mutations might be found in lymphoma cases where JAK3 is reported to be activated. We, therefore, sequenced LNK PH domain in 147 lymphoma samples including- mantle cell lymphoma, peripheral T cell lymphoma (PTCL) and adult T cell leukemia/lymphoma cases but could not detect any mutations. In summary- we found that JAK3 binds and phosphorylates both WT-LNK and to a greater extent, the LNK PH domain mutant E208Q and in the absence of a cytokine receptor can lead to JAK3 phosphorylation. Taken together, LNK may likely control JAK3 activity. Disclosures: No relevant conflicts of interest to declare.