14-3-3 Regulates the Lnk/JAK2 Pathway In Hematopoietic Stem and Progenitor Cells

Abstract 86

Hematopoietic stem and progenitor cell (HSPC) homeostasis is regulated by intricate signaling networks. The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis and its dysregulation can lead to hematologic malignancies. Recently activating mutations in JAK2 were found in a large fraction of patients with myeloproliferative neoplasms (MPNs). We previously demonstrated that lymphocyte adaptor protein (Lnk) binds JAK2 and attenuates its activity, thereby limiting HSPC expansion (Bersenev et al., JCI, 2008;118:2832-2844). We further showed that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPNs in mice (Bersenev et al., JCI, 2010;120:2058-2069). Lnk directly inhibits oncogenic JAK2 as well as acting through JAK2- independent pathways to constrain MPN development. Consistently, aged Lnk^–/– mice spontaneously developed a CML-like MPN (Bersenev et al., JCI, 2010;120:2058-2069). More importantly, loss-of-function mutations in Lnk are found in human MPN patients (Oh, et. al., Blood, 2010, in press). This work suggests that Lnk plays a pivotal role in regulating both normal and malignant HSPC expansion. However, it remains to be determined how Lnk attenuates JAK2 activity since Lnk itself does not possess any enzymatic activity. Therefore, we began exploring Lnk regulatory mechanisms by identifying novel Lnk partners through protein purification and mass spectrometric analysis. This resulted in the identification of a number of novel Lnk binding proteins, which include the 14-3-3 proteins as the most robust interactors. 14-3-3s are a group of scaffold proteins that regulate many disease-relevant gene products and play important functions in many aspects of cellular processes. We found that Lnk is phosphorylated at two serine residues, which serve as the critical binding sites for 14-3-3. 14-3-3 abrogates the Lnk-JAK2 interaction thereby alleviating Lnk inhibitory function in both JAK2 signaling and cell growth. Furthermore, 14-3-3 binding is necessary and sufficient to maintain Lnk in an inactive state. We also investigated the signals that regulate Lnk phosphorylation, revealing Lnk as a signaling nodal point that integrates multiple signaling pathways in controlling HSPC homeostasis. The physiological significance of the Lnk-14-3-3 interaction in HSPC development will be discussed. Thus, our data implicate previously unappreciated serine phosphorylation events in Lnk-dependent hematopoietic function and regulation of JAK2. In addition, identification of novel signaling molecules that influence hematopoiesis might facilitate stem cell therapies and provide novel therapeutic targets for the treatment of MPNs.