Related Experiment Videos
Repression of c-Cbl leads to enhanced G-CSF Jak-STAT signaling without increased cell proliferation
Lin Wang1, William A Rudert, Inna Loutaev
1Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Abstract:
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.
Insights
Granulocyte-colony-stimulating factor (G-CSF) signaling relies on c-Cbl for growth. While c-Cbl deficiency enhances Jak-STAT activation, it impairs G-CSF-induced proliferation, highlighting c-Cbl
Area of Science:
- Cellular signaling pathways
- Hematopoiesis and immunology
- Molecular biology
Background:
- Granulocyte-colony-stimulating factor (G-CSF) receptor activation initiates signaling cascades involving Lyn and Jak2 tyrosine kinases.
- Lyn kinase is crucial for G-CSF-induced mitogenic signaling, as demonstrated by its absence in Lyn-deficient DT40 cells.
- c-Cbl is a key adaptor protein in hematopoietic cells, mediating cell growth and cytoskeletal dynamics.
Purpose of the Study:
- To elucidate the role of c-Cbl in G-CSF receptor-mediated signaling pathways.
- To investigate the interaction between Lyn, c-Cbl, and PI 3-kinase in G-CSF signaling.
- To determine the contribution of c-Cbl to G-CSF-induced proliferation and DNA synthesis.
Main Methods:
- Utilized Lyn-deficient DT40 cells expressing the G-CSF receptor (DT40GR) to study G-CSF signaling.
- Employed yeast two-hybrid analysis to identify direct interactions between c-Cbl and Lyn.
- Constructed and utilized antisense vectors for c-Cbl inhibition and analyzed G-CSF-induced signaling, PI 3-kinase activity, and DNA synthesis.
Main Results:
- Lyn associates with and phosphorylates c-Cbl, which directly couples Lyn to PI 3-kinase.
- A c-Cbl mutant (Y731F) that uncouples PI 3-kinase inhibits G-CSF-induced proliferation.
- Antisense inhibition of c-Cbl enhances Jak-STAT activation but decreases G-CSF-induced PI 3-kinase activity and DNA synthesis.
Conclusions:
- Physiological levels of c-Cbl are essential for a G-CSF-driven growth stimulatory pathway.
- Enhanced Jak-STAT activation alone is insufficient to drive G-CSF-induced cellular proliferation.
- c-Cbl acts as a critical mediator linking G-CSF receptor engagement to proliferative signaling via PI 3-kinase.