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 9

HSPC reconstitution in mice requires 14-3-3 proteins.

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HSPC reconstitution in mice requires 14-3-3 proteins. (A) Lin– BM cells ...
(A) Lin– BM cells were infected with pLKO-shRNA lentiviruses to either Luc or 14-3-3 ε, β, or ζ. Knockdown efficiencies were examined by WB. Asterisks indicate clones with high knockdown efficiencies. All the lanes were run on the same gel but were noncontiguous. No., indicates clone number. (B) Infected Lin– BM cells were transplanted into lethally irradiated recipient mice. The percentages of GFP+ reconstitutions in the PB of host animals 4, 8, and 12 weeks after BMT are shown. Time 0 indicates time of transplant. Each line represents an individual transplanted animal. (C–F) Pan 14-3-3 knockdown inhibits HSPC reconstitution. (C) Lin– BM cells were infected with pLKO-shRNA lentiviruses to either Luc or 2 pan 14-3-3 clones (no. 1 or no. 2). Knockdown efficiencies were examined by WB. All the lanes were run on the same gel but were noncontiguous. (D) Lin– BM cells expressing shRNAs were transplanted. FACS plots of the percentages of GFP+ in donor BM cells before transplant and host PB 16 weeks after transplant are shown. Results are pooled from 2 independent experiments. *P < 0.0001, **P < 0.01, compared with Luc control. (E) The percentage of GFP+ reconstitutions in donor-derived PB of host animals at indicated times is shown. (F) The percentage of GFP+reconstitutions in total PB of host animals at 12 weeks is shown (n = 6–10). Average ± SEM.