Longva Karianne E, Blystad Froydis D, Stang Espen, Larsen Astrid M, Johannessen Lene E, Madshus Inger H
Institute of Pathology, The University of Oslo, Rikshospitalet, N-0027 Oslo, Norway.
J Cell Biol. 2002 Mar 4;156(5):843-54. doi: 10.1083/jcb.200106056. Epub 2002 Feb 25.
EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.
表皮生长因子（EGF）而非转化生长因子α（TGFα）能有效地诱导表皮生长因子受体（EGFR）的降解。我们发现，在与EGF和TGFα孵育时，EGFR最初被多聚泛素化的程度相同，然而与TGFα相比，与EGF孵育时泛素化更持久。一致地，在用EGF和TGFα激活EGFR后，泛素连接酶c-Cbl被募集到质膜，但仅在用EGF激活时定位于内体。内吞作用后EGF仍与EGFR结合，而TGFα则从EGFR上解离。因此，持续的多聚泛素化是由EGF确保内吞的EGFR的激酶活性来解释的。显性负性N-Cbl的过表达抑制了EGFR的泛素化以及EGF和EGFR的降解。这表明EGF诱导的EGFR泛素化本身对于溶酶体分选很重要。溶酶体和蛋白酶体抑制剂均阻断了EGF和EGFR的降解，并且蛋白酶体抑制剂抑制了活化的EGFR从多囊泡体（MVB）的外限制膜向内膜的转运。因此，激酶活性EGFR的溶酶体分选受蛋白酶体活性调节。免疫电子显微镜显示完整的EGFR定位于MVB的内膜上。这表明EGFR本身不是蛋白酶体的靶标。
