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 7

Lnk binds to JAK2 Y613 and Y813 residues in response to TPO.

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The Lnk SH2 domain associates with kinase-active JAK2. (A) Stable 32D-B/...
(A) We established stable 32D-B/A cell lines expressing Flag-Lnk and Mpl along with myc-tagged WT JAK2 or various JAK2 mutants. Cells were starved and stimulated with TPO. Top: IPs with Flag-specific antibodies and blots sequentially probed with antibodies specific for myc, JAK2, and Lnk. Bottom: IPs with myc-specific antibodies and blots sequentially probed with 4G10-specific and JAK2-specific antibodies. C, control 32D cells expressing Lnk alone. (B) Top: Flag-Lnk and either vector alone or various myc-JAK2 constructs were transiently transfected into 293T cells. Lysates were precipitated with Flag-specific antibodies and sequentially probed with antibodies specific for myc, 4G10, and Lnk. Bottom: Cell lysates were precipitated with myc-specific antibodies and probed with 4G10- or JAK2-specific antibodies. (C) Stable 32D-B/A cell lines expressing Flag-Lnk and Mpl, along with vector alone or myc-tagged WT JAK2 or JAK2 V617F (J2V617F) mutant were starved and stimulated with TPO. Top: IPs with Flag-specific antibodies and blots with antibodies specific for myc, JAK2, and Lnk. Bottom: IPs with myc-specific antibodies and blots with 4G10- and JAK2-specific antibodies.