Brief communicationGenetic disruption of the scaffolding protein, Kinase Suppressor of Ras 1 (KSR1), differentially regulates GM-CSF-stimulated hyperproliferation in hematopoietic progenitors expressing activating PTPN11 mutants D61Y and E76K
Introduction
Juvenile myelomonocytic leukemia (JMML) is a lethal childhood disease clinically characterized by overproduction of myelomonocytic cells and by the in vitro phenotype of hematopoietic progenitor hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). At the molecular level, Ras hyperactivation is implicated based on the majority of JMML patients bearing either loss-of-function NF1 mutations or gain-of-function RAS or PTPN11 mutations [1]. We demonstrated previously that the PTPN11 gain-of-function mutants Shp2E76K and Shp2D61Y induce constitutively elevated and sustained activation of Erk [2], verifying the relevance the Ras-mitogen activated protein kinase (Raf1/MEK/Erk) cascade in this disease.
Signal transduction among Raf1/MEK/Erk kinases is mediated through direct phosphorylation, but scaffolding proteins also play an important role in regulating the location, strength, and duration of Raf1/MEK/Erk signaling. One of the best-defined scaffolding proteins that positively facilitates the Raf1/MEK/Erk cascade is Kinase Suppressor of Ras (KSR1) [3]. In quiescent cells, KSR1 is phosphorylated and constitutively associated with MEK. In response to growth factor stimulation or Ras activation, KSR1 is dephosphorylated (serine 392), translocates to the cell membrane, co-localizes MEK with Raf1 and Erk, and promotes Erk activation [4]. Inhibition and/or genetic deletion of KSR1 have been demonstrated to reduce the capacity of gain-of-function Ras-induced tumorigenesis [5], [6]. Based on our previous observation of constitutively activated Erk in Shp2E76K- and Shp2D61Y-expressing cells, we hypothesized that KSR1 may contribute to the hyperproliferation and GM-CSF hypersensitivity of mutant Shp2-expressing cells.
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Mice and cells
Generation of mice bearing genetic disruption of KSR1 has been previously described [6]. KSR1+/− mice were crossed to generate KSR1−/− progeny and WT (KSR1+/+) littermate controls. Progeny from these crosses were genotyped by polymerase chain reaction (PCR), as previously described [6]. All animal studies were approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine. Bone marrow low density mononuclear cells (LDMNCs) were either plated into
KSR1 activation and interaction with phospho-Erk is increased in Shp2D61Y- and Shp2E76K-expressing macrophage progenitors
Previously, we demonstrated hyperactivation of GM-CSF-stimulated Erk activation in macrophage progenitors expressing the Shp2 gain-of-function mutants, D61Y and E76K [2]. Following GM-CSF stimulation, decreased levels of phospho-KSR1 were found in cells expressing Shp2 gain-of-function mutants compared to cells transduced with empty vector or WT Shp2, demonstrating enhanced KSR1 activation in the mutant Shp2-expressing cells (Fig. 1). Consistently, significantly higher levels of phospho-Erk
Discussion
By regulating Raf1/MEK/Erk signaling, KSR1 is involved in many fundamental cellular processes including inhibition of tumor development [5], [6], [8]; thus, we predicted that KSR1 contributes to the regulation of GM-CSF-induced hyperactivation of Erk and hyperproliferation in mutant Shp2-expressing progenitors. This prediction was supported by our biochemical findings demonstrating increased KSR1 activation and association with phospho-Erk in Shp2D61Y- and Shp2E76K-expressing macrophage
Contributors
ZY – designed/performed research, wrote paper; MC, SAS, TS, and FY – designed and performed research; MY – performed statistical analysis; FCY and RJC designed research and wrote paper.
Conflict of interest
The authors have no conflicting financial interests.
Acknowledgements
This work was supported by the U.S. National Institutes of Health (HL082981, RJC) and the U.S. Department of Defense (DOD W81XWH-05-1-0161 and DOD W81XWH-08-1-0071, FCY). The authors gratefully acknowledge the administrative assistance of Linda S. Henson.
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2018, Stem Cells International