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.
Highlights
Identified in the past few years, including phospholipase C-␥ [4, 5], the guanine nucleotide exchange factor Vav (6 – 8), an oligomeric ATPase valosin-containing protein [9, 10], the membrane-cytoskeleton linker protein ezrin [11, 12], and the subunit of T cell antigen receptor (TCR) [13]
Tyrosine Phosphorylation in protein kinase C (PKC)-activated Cells ester-stimulated IL-2 mRNA production [40], which raised the possibility that tyrosine phosphorylation may be important for phorbol ester-stimulated IL-2 production
C-Cbl proteins were immunoprecipitated from both S- and resistant EL4 (R-EL4) cells, and the tyrosine phosphorylation of c-Cbl was determined by anti-Tyr(P) immunoblotting
Summary
Identified in the past few years, including phospholipase C-␥ [4, 5], the guanine nucleotide exchange factor Vav (6 – 8), an oligomeric ATPase valosin-containing protein [9, 10], the membrane-cytoskeleton linker protein ezrin [11, 12], and the subunit of TCR [13]. C-Cbl proteins were immunoprecipitated from lysates of cells that had been unstimulated or treated with PDB for 2 min, and Cbl-associated tyrosine phosphoproteins were detected with an anti-Tyr(P) antibody.
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