Repression of c-Cbl leads to enhanced G-CSF Jak-STAT signaling without increased cell proliferation.
Engagement of the Granulocyte-Colony-Stimulating Factor (G-CSF) receptor activates non-receptor protein tyrosine kinases Lyn and Jak2. We found that Lyn-deficient DT40 cells that express the G-CSF receptor (DT40GR) do not demonstrate G-CSF-induced mitogenic signaling. Lyn associates with and phosphorylates a small set of molecules, including c-Cbl. c-Cbl is an adaptor involved in cell growth and cytoskeletal reorganization, predominantly in hematopoietic cells. Using yeast two-hybrid analysis, we found that c-Cbl directly couples Lyn to PI 3-kinase. We also found that expression of the c-CblY731F mutant, which uncouples PI 3-kinase, resulted in the inhibition of G-CSF-induced proliferative signaling in DT40GR cells. As a complementary strategy, we sought to analyse the effects of c-Cbl deficiency in DT40GR cells. We isolated, cloned and sequenced the full-length cDNA for chicken c-Cbl and constructed antisense vectors. Antisense inhibition of c-Cbl expression in DT40GR cells led to enhanced Jak-STAT activation following G-CSF stimulation. Yet, this enhancement of Jak-STAT activation was associated with decreased G-CSF-induced PI 3-kinase activity and DNA synthesis. PI 3-kinase activity correlated with DNA synthesis and physiological levels of c-Cbl. Together, these data suggest that physiologic level of c-Cbl provides a growth stimulatory pathway for G-CSF and that enhanced Jak-STAT activation is not sufficient for G-CSF-induced growth.
Wang
Lin
L
Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Rudert
William A
WA
Loutaev
Inna
I
Roginskaya
Vera
V
Corey
Seth J
SJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D017173:Caenorhabditis elegans; D002455:Cell Division; D002645:Chickens; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D053614:Janus Kinase 2; D051379:Mice; D008894:Milk Proteins; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D019061:src-Family Kinases / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D016188:Receptors, Granulocyte Colony-Stimulating Factor; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; C494080:Stat1 protein, mouse; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D016179:Granulocyte Colony-Stimulating Factor; D006160:Guanosine Triphosphate; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C067419:CBL protein, human; C493849:Cbl protein, mouse