NVP-BKM120 PI3K inhibitor we investigated the interaction between the Cbl proteins and the EGFRvIII further.

e Cbl proteins. Therefore, we investigated the interaction between the Cbl proteins and the EGFRvIII further. In contrast to the published data, we found that the overexpression of all three Cbl proteins NVP-BKM120 PI3K inhibitor caused the Davies et al. Page 2 Oncogene. Author manuscript, available in PMC 2008 March 25. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript ubiquitination and downregulation of the EGFRvIII. Also, we demonstrated that Cbl b binds to the EGFRvIII, that the requirements for Cbl b mediated degradation of the EGFRvIII are identical to that of the WT EGFR, and that only active EGFRvIII is downregulated. Consequently, Cbl b inhibits the transformation of NIH 3T3 fibroblasts by the EGFRvIII and the mutation of the Cbl binding site in the EGFRvIII enhances the ability of the EGFRvIII to transform.
Finally, we demonstrated that the inhibition of the EGFRvIII TK by AG 1478, which abrogates Cbl mediated downregulation of the EGFR vIII, antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. Thus, the EGFRvIII undergoes activation dependent downregulation mediated by the Cbl proteins. Results Bosutinib 380843-75-4 Cbl proteins ubiquitinate and downregulate the constitutively active EGFRvIII The overexpression of Cbl proteins enhances EGF induced ubiquitination and downregulation of the WT EGFR. Therefore, we investigated whether the Cbl proteins also regulate the constitutively active mutant EGFRvIII in a cell line Chinese hamster ovary that does not express the WT EGFR.
The co transfection of CHO cells with the EGFRvIII and either Cbl, Cbl b, or Cbl c resulted in a decrease in EGFRvIII protein levels. Also, the co transfection of Cbl, Cbl b, or Cbl c increased the amount of ubiquitinated proteins seen in immunoprecipitates of the EGFRvIII. These ubiquitinated species represent ubiquitinated forms of the EGFRvIII suggesting that, like the active WT EGFR, Cbl proteins are capable of ubiquitinating and downregulating the EGFRvIII. As all three Cbl proteins caused the degradation of the EGFRvIII, we chose to use Cbl b to investigate the mechanism by which they regulate this oncogenic EGFR mutant. Given that the TK activity and autophosphorylation of the WT EGFR are necessary for its ubiquitination and degradation by the Cbl proteins, we examined whether this is also the case with the EGFRvIII.
Although the WT EGFR is regulated by ligand binding, the EGFRvIII is spontaneously active. Therefore, we used the EGFR TK inhibitor AG 1478 to inhibit the activity of the EGFRvIII. Treatment of CHO cells overexpressing the EGFRvIII with AG 1478 prevented tyrosine autophosphorylation of the EGFRvIII. Inactivation of the EGFRvIII TK by AG 1478 attenuated its downregulation by Cbl b. Co expression of Cbl b resulted in downregulation of the EGFRvIII by 73% in the absence of AG 1478. In the presense of AG 1478, the level of the EGFRvIII was higher and co expression of Cbl b only resulted in 5% downregulation. AG 1478 completely abolished ubiquitination of EGFRvIII by Cbl b. Also, AG 1478 treatment inhibited the ubiquitination and downregulation of the EGFRvIII by Cbl. Therefore, the TK activity of the EGFRvIII is necessary for its downregulation by the Cbl proteins. As AG 1478 inhibits the activation induced downregulation of the EGFRvIII by the Cbl proteins, we examined the effects of AG 1478 upon the subce