CBL linker region and RING finger mutations lead to enhanced granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling via elevated levels of JAK2 and LYN

J Biol Chem. 2013 Jul 5;288(27):19459-70. doi: 10.1074/jbc.M113.475087. Epub 2013 May 21.


Juvenile myelomonocytic leukemia (JMML) is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). SHP2, NF-1, KRAS, and NRAS are mutated in JMML patients, leading to aberrant regulation of RAS signaling. A subset of JMML patients harbor CBL mutations associated with 11q acquired uniparental disomy. Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. We investigated the mechanism by which CBL-Y371H, a linker region mutant, and CBL-C384R, a RING finger mutant, lead to enhanced GM-CSF signaling. Expression of CBL mutants in the TF-1 cell line resulted in enhanced survival in the absence of GM-CSF. Cells expressing CBL mutations displayed increased phosphorylation of GM-CSF receptor βc subunit in response to stimulation, although expression of total GM-CSFR βc was lower. This suggested enhanced kinase activity downstream of GM-CSFR. JAK2 and LYN kinase expression is elevated in CBL-Y371H and CBL-C384R mutant cells, resulting in enhanced phosphorylation of CBL and S6 in response to GM-CSF stimulation. Incubation with the JAK2 inhibitor, TG101348, abolished the increased phosphorylation of GM-CSFR βc in cells expressing CBL mutants, whereas treatment with the SRC kinase inhibitor dasatinib resulted in equalization of GM-CSFR βc phosphorylation signal between wild type CBL and CBL mutant samples. Dasatinib treatment inhibited the elevated phosphorylation of CBL-Y371H and CBL-C384R mutants. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signaling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways.

Keywords: CBL; Cytokine; E3 Ubiquitin Ligase; GM-CSF; JAK Kinase; Juvenile Myelomonocytic Leukemia; SRC; Signal Transduction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Cell Line
  • Cytokine Receptor Common beta Subunit / genetics
  • Cytokine Receptor Common beta Subunit / metabolism*
  • Dasatinib
  • Gene Expression Regulation, Enzymologic*
  • Gene Expression Regulation, Leukemic / drug effects
  • Gene Expression Regulation, Leukemic / genetics
  • Granulocyte-Macrophage Colony-Stimulating Factor / genetics
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism*
  • Humans
  • Janus Kinase 2 / antagonists & inhibitors
  • Janus Kinase 2 / genetics
  • Janus Kinase 2 / metabolism*
  • Leukemia, Myelomonocytic, Juvenile / genetics
  • Leukemia, Myelomonocytic, Juvenile / metabolism
  • Leukemia, Myelomonocytic, Juvenile / pathology
  • Mutation, Missense*
  • Phosphorylation / drug effects
  • Phosphorylation / genetics
  • Protein Kinase Inhibitors / pharmacology
  • Proto-Oncogene Proteins c-cbl / biosynthesis*
  • Proto-Oncogene Proteins c-cbl / genetics
  • Pyrimidines / pharmacology
  • Pyrrolidines / pharmacology
  • RING Finger Domains / genetics
  • Signal Transduction*
  • Sulfonamides / pharmacology
  • Thiazoles / pharmacology
  • src-Family Kinases / biosynthesis*
  • src-Family Kinases / genetics


  • Cytokine Receptor Common beta Subunit
  • Protein Kinase Inhibitors
  • Pyrimidines
  • Pyrrolidines
  • Sulfonamides
  • Thiazoles
  • fedratinib
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Proto-Oncogene Proteins c-cbl
  • JAK2 protein, human
  • Janus Kinase 2
  • lyn protein-tyrosine kinase
  • src-Family Kinases
  • CBL protein, human
  • Dasatinib