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Deficient tyrosine phosphorylation of c-Cbl and associated proteins in phorbol ester-resistant EL4 mouse thymoma
1Department of Pharmacology and Cancer Center, University of Virginia, Charlottesville, Virginia 22908, USA.
Abstract:
Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.
Insights
Phorbol ester stimulation enhances tyrosine phosphorylation of c-Cbl and phosphatidylinositol 3-kinase (PI3K) p85 subunit in EL4 cells. Impaired phosphorylation in resistant cells suggests a role in phorbol ester response.
Area of Science:
- Cellular signaling pathways
- Molecular biology
- Oncogenesis
Background:
- Phorbol esters are potent activators of protein kinase C.
- EL4 mouse thymoma cells are utilized to study cellular responses to stimuli.
- c-Cbl protooncogene and phosphatidylinositol 3-kinase (PI3K) are key signaling molecules.
Purpose of the Study:
- To investigate the role of c-Cbl and PI3K p85 subunit tyrosine phosphorylation in phorbol ester-stimulated EL4 cells.
- To compare signaling events in phorbol ester-sensitive (S-EL4) and resistant (R-EL4) EL4 cells.
Main Methods:
- Co-immunoprecipitation to identify protein complexes.
- In vitro binding assays to assess protein interactions.
- In vitro kinase assays to determine phosphorylation activity.
Main Results:
- Tyrosine phosphorylation of c-Cbl and PI3K p85 increased upon phorbol ester stimulation in S-EL4 cells.
- R-EL4 cells showed diminished tyrosine phosphorylation of c-Cbl and p85.
- Complexes of c-Cbl with p85, and p85 with Lck tyrosine kinase were observed in stimulated S-EL4 cells.
- Lck phosphorylates p85 in stimulated S-EL4 cells, forming a ternary complex.
Conclusions:
- Tyrosine phosphorylation of c-Cbl and PI3K p85 is crucial for phorbol ester response in EL4 cells.
- Defective phosphorylation in R-EL4 cells contributes to their resistance to phorbol esters.
- These findings elucidate a signaling mechanism involving c-Cbl, PI3K, and Lck in T-cell activation.
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