TY - JOUR
T1 - EGF receptor ubiquitination is not necessary for its internalization
AU - Huang, Fangtian
AU - Lai, Kuan Goh
AU - Sorkin, Alexander
PY - 2007/10/23
Y1 - 2007/10/23
N2 - Ubiquitination of the EGF receptor (EGFR) has been implicated in EGF-induced receptor internalization, lysosomal degradation, and down-regulation. Mutation of EGFR ubiquitination sites identified by mass spectrometry yielded receptor mutants that are weakly ubiquitinated and not down-regulated by EGF. However, these EGFR mutants were normally internalized. To examine whether this internalization was mediated by the residual ubiquitination, systematic mutagenesis of lysine residues in the kinase domain of the EGFR was performed to generate a receptor mutant that is not ubiquitinated. Mutations of a number of lysines inhibited kinase activity of the EGFR, thus leading to the inhibition of receptor internalization. However, a mutant lacking 15 lysine residues (15KR), which was negligibly ubiquitinated and normally phosphorylated, was internalized at a rate similar to that of the wild-type EGFR. As in the case of the wild-type EGFR, internalization of the 15KR mutant depended on the presence of clathrin, Grb2 adaptor, and Cbl ubiquitin ligase. These data imply that EGFR ubiquitination is not necessary for its internalization by clathrincoated pits. Interestingly, the reconstitution of two major ubiquitination sites in the 16KR receptor mutant, which had impaired kinase activity and slow internalization kinetics, resulted in a partial rescue of ubiquitination and a complete rescue of receptor internalization. This result suggests that ubiquitination of the kinase-impaired receptor can mediate its internalization by the clathrin pathway. Altogether these data emphasize the robustness of the EGFR internalization process, which can be controlled by multiple kinase- and ubiquitination-dependent and -independent mechanisms.
AB - Ubiquitination of the EGF receptor (EGFR) has been implicated in EGF-induced receptor internalization, lysosomal degradation, and down-regulation. Mutation of EGFR ubiquitination sites identified by mass spectrometry yielded receptor mutants that are weakly ubiquitinated and not down-regulated by EGF. However, these EGFR mutants were normally internalized. To examine whether this internalization was mediated by the residual ubiquitination, systematic mutagenesis of lysine residues in the kinase domain of the EGFR was performed to generate a receptor mutant that is not ubiquitinated. Mutations of a number of lysines inhibited kinase activity of the EGFR, thus leading to the inhibition of receptor internalization. However, a mutant lacking 15 lysine residues (15KR), which was negligibly ubiquitinated and normally phosphorylated, was internalized at a rate similar to that of the wild-type EGFR. As in the case of the wild-type EGFR, internalization of the 15KR mutant depended on the presence of clathrin, Grb2 adaptor, and Cbl ubiquitin ligase. These data imply that EGFR ubiquitination is not necessary for its internalization by clathrincoated pits. Interestingly, the reconstitution of two major ubiquitination sites in the 16KR receptor mutant, which had impaired kinase activity and slow internalization kinetics, resulted in a partial rescue of ubiquitination and a complete rescue of receptor internalization. This result suggests that ubiquitination of the kinase-impaired receptor can mediate its internalization by the clathrin pathway. Altogether these data emphasize the robustness of the EGFR internalization process, which can be controlled by multiple kinase- and ubiquitination-dependent and -independent mechanisms.
KW - Clathrin
KW - Endocytosis
KW - Ubiquitin
UR - http://www.scopus.com/inward/record.url?scp=36749036679&partnerID=8YFLogxK
U2 - 10.1073/pnas.0707416104
DO - 10.1073/pnas.0707416104
M3 - Article
C2 - 17940017
AN - SCOPUS:36749036679
SN - 0027-8424
VL - 104
SP - 16904
EP - 16909
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 43
ER -