14-3-3 regulates the LNK/JAK2 pathway in mouse hematopoietic stem and progenitor cells
Jing Jiang, … , Yiwen Song, Wei Tong
Jing Jiang, … , Yiwen Song, Wei Tong
Published May 1, 2012
Citation Information: J Clin Invest. 2012;122(6):2079-2091. https://doi.org/10.1172/JCI59719.
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Research Article Hematology

14-3-3 regulates the LNK/JAK2 pathway in mouse hematopoietic stem and progenitor cells

Abstract

Hematopoietic stem and progenitor cell (HSPC) functions are governed by intricate signaling networks. The tyrosine kinase JAK2 plays an essential role in cytokine signaling during hematopoiesis. The adaptor protein LNK is a critical determinant of this process through its inhibitory interaction with JAK2, thereby limiting HSPC self-renewal. LNK deficiency promotes myeloproliferative neoplasm (MPN) development in mice, and LNK loss-of-function mutations are found in human MPNs, emphasizing its pivotal role in normal and malignant HSPCs. Here, we report the identification of 14-3-3 proteins as LNK binding partners. 14-3-3 interfered with the LNK-JAK2 interaction, thereby alleviating LNK inhibition of JAK2 signaling and cell proliferation. Binding of 14-3-3 required 2 previously unappreciated serine phosphorylation sites in LNK, and we found that their phosphorylation is mediated by glycogen synthase kinase 3 and PKA kinases. Mutations of these residues abrogated the interaction and augmented the growth inhibitory function of LNK. Conversely, forced 14-3-3 binding constrained LNK function. Furthermore, interaction with 14-3-3 sequestered LNK in the cytoplasm away from the plasma membrane-proximal JAK2. Importantly, bone marrow transplantation studies revealed an essential role for 14-3-3 in HSPC reconstitution that can be partially mitigated by LNK deficiency. We believe that, together, this work implicates 14-3-3 proteins as novel and positive HSPC regulators by impinging on the LNK/JAK2 pathway.

Authors

Jing Jiang, Joanna Balcerek, Krasimira Rozenova, Ying Cheng, Alexey Bersenev, Chao Wu, Yiwen Song, Wei Tong

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Figure 1

Identification of 14-3-3 proteins as LNK binding partners.

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Identification of 14-3-3 proteins as LNK binding partners. (A) Cytoplasm...
(A) Cytoplasmic fractions of 32D-B/A parental cells (C) or cells stably expressing Flag/HA-LNK (LNK) were isolated and precipitated with anti-Flag (1°) and anti-HA antibodies (2°) sequentially. IP eluates were subjected to SDS-PAGE and visualized by silver staining. M, marker. (B) LNK interacts with endogenous 14-3-3. Protein lysates from WT and Lnk–/– spleens were either directly subjected to WB analysis or IP with anti-LNK antibodies, followed by WB analysis with indicated antibodies. ns, nonspecific. (C) 32D-B/A cells stably expressing Lnk were stimulated with or without Tpo for 10 minutes. Cell lysates were subjected to WB analysis with phospho-specific antibodies to pS13 and pS129 sites in LNK or total LNK protein.