Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2
Alexey Bersenev, Chao Wu, Joanna Balcerek, Wei Tong
Alexey Bersenev, Chao Wu, Joanna Balcerek, Wei Tong
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Research Article Hematology

Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2

Abstract

In addition to its role in megakaryocyte production, signaling initiated by thrombopoietin (TPO) activation of its receptor, myeloproliferative leukemia virus protooncogene (c-Mpl, or Mpl), controls HSC homeostasis and self-renewal. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. We found that HSCs from Lnk–/– mice have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeat treatments with cytoablative 5-fluorouracil in vivo compared with WT HSCs. We further provide genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal, predominantly through Mpl. Consistent with this observation, Lnk–/– HSCs displayed potentiated activation of JAK2 specifically in response to TPO. Biochemical experiments revealed that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Of note, the JAK2 V617F mutant, found at high frequencies in myeloproliferative diseases, retains the ability to bind Lnk. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells and TPO/Mpl/JAK2/Lnk as a major regulatory pathway in controlling stem cell self-renewal and quiescence.

Authors

Alexey Bersenev, Chao Wu, Joanna Balcerek, Wei Tong

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Figure 6

Lnk constitutively associates Mpl.

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Lnk binds to JAK2 Y613 and Y813 residues in response to TPO. (A) We esta...
We established stable 32D-B/A cell lines expressing Flag-Lnk along with WT Mpl or various Mpl mutants. Cells were starved and then stimulated with TPO. (A) Left: Lysates from cells expressing either WT Mpl or the Y5F mutant were precipitated with Flag-specific antibodies and sequentially probed with antibodies specific for JAK2, Mpl, 4G10, and Lnk. Right: Cell lysates were also reciprocally precipitated with Mpl-specific antibodies and sequentially probed with antibodies specific for Flag, 4G10, and HA. (B) Left: Lysates from cells expressing WT Mpl or the Mpl mutants, Box1 or LW, were precipitated with Flag-specific antibodies and sequentially probed with antibodies specific for JAK2, Mpl, and Lnk. Right: Cell lysates were precipitated with Mpl-specific or JAK2-specific antibodies and probed with 4G10-specific antibodies, followed by antibodies specific for total Mpl and JAK2.