llular localization BMS-536924 IGF-1R inhibitor of the EGFRvIII in the murine fibroblast cell line NR 6m. The NR 6m cell line is a variant of Swiss 3T3 cells which has been stably transfected with the EGFRvIII, resulting in transformation of the cells. It was chosen for the localization studies because the cell line does not express endogenous WT EGFR, thus allowing the use of anti EGFR and anti phospho EGFR antibodies. In these cells, the EGFRvIII was localized in both the plasma membrane and intracellular vesicles. The majority of active EGFRvIII, as detected by EGFR phosphotyrosine 1173 staining, appears to be localized in intracellular vesicles. Inhibition of the TK activity of Davies et al. Page 3 Oncogene. Author manuscript, available in PMC 2008 March 25.
NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript the EGFRvIII by AG 1478 treatment abolished phosphotyrosine 1173 staining and resulted in a reduction of the amount of EGFRvIII in intracellular vesicles and an increase in the proportion of the EGFRvIII located at the plasma membrane compared to intracellular vesicles. This is consistent with AG 1478 treatment SKI-606 380843-75-4 preventing activation induced internalization and downregulation of the EGFRvIII from the plasma membrane. Requirements for Cbl b mediated downregulation of the EGFRvIII We mapped the regions of Cbl b necessary for the downregulation of the EGFRvIII by transfecting CHO cells with the EGFRvIII and various constructs of Cbl b. As described above, WT Cbl b downregulates the EGFRvIII. The deletion of the proline rich, carboxy terminal half of Cbl b did not inhibit its ability to downregulate the EGFRvIII.
In contrast, the deletion of the TKB domain containing the aminoterminus of Cbl b prevented the downregulation of the EGFRvIII by Cbl b. Finally, a RING finger mutant of Cbl b that has been shown to lack E3 activity was unable to downregulate the EGFRvIII. Quantification of the downregulation of the EGFRvIII by the various constructs of Cbl b revealed that N1/2 and WT Cbl b downregulate the EGFRvIII to a similar extent, that the overexpression of C2/3 Cbl b did not affect EGFRvIII levels, and that the RING finger mutant of Cbl b tended to increase the amount of the EGFRvIII protein. Therefore, like the WT EGFR, the TKB and RING finger domains of Cbl b are sufficient for the downregulation of the EGFRvIII.
Also, the E3 activity of Cbl b is necessary for the downregulation of the EGFRvIII by Cbl b. The TKB domain of the Cbl proteins has been shown to mediate a specific binding to a phosphotyrosine residue in the activated WT EGFR. The mutation of this residue attenuates the downregulation of the EGFR. We tested the ability of the equivalent mutation in the EGFRvIII to affect its regulation by Cbl b. Using an antibody against phosphotyrosine 1045 EGFR, we detected phosphorylation of the EGFRvIII at this residue that was abolished by its mutation to phenylalanine. As in the WT EGFR, Y1045 appears to be a minor phosphotyrosine residue, as the loss of Y1045 phosphorylation by mutation of this residue does not decrease significantly the content of EGFRvIII phosphotyrosine. As described above, the EGFRvIII is ubiquitinated and downregulated by both WT and N1/2 Cbl b. In contrast, the Y1045F mutation in the EGFRvIII abolishes the ability of N1/2, but not WT Cbl b to ubiquitinate the EGFRvIII. This mutation also attenuates the downregulatio
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