Differential regulation of EGF receptor internalization and degradation by multiubiquitination within the kinase domain.
Ubiquitination of the EGF receptor (EGFR) is believed to play a critical role in regulating both its localization and its stability. To elucidate the role of EGFR ubiquitination, tandem mass spectrometry was used to identify six distinct lysine residues within the kinase domain of the EGFR, which can be conjugated to ubiquitin following growth factor stimulation. Substitution of these lysine residues with arginines resulted in a dramatic decrease in overall ubiquitination but preserved normal tyrosine phosphorylation of EGFR. Ubiquitination-deficient EGFR mutants displayed a severe defect in their turnover rates but were internalized at rates comparable to those of wild-type receptors. Finally, quantitative mass spectrometry demonstrated that more than 50% of all EGFR bound ubiquitin was in the form of polyubiquitin chains, primarily linked through Lys63. Taken together, these data provide direct evidence for the role of EGFR ubiquitination in receptor targeting to the lysosome and implicate Lys63-linked polyubiquitin chains in this sorting process.
Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, 80045, USA.
Fangtian
F
Huang
Donald
D
Kirkpatrick
Xuejun
X
Jiang
Steven
S
Gygi
Alexander
A
Sorkin
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004705:Endocytosis; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D051380:GRB2 Adaptor Protein / Q000502:physiology; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D050721:Proto-Oncogene Proteins c-cbl / Q000502:physiology; D015398:Signal Transduction; D013552:Swine; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
D051380:GRB2 Adaptor Protein; D025801:Ubiquitin; D050721:Proto-Oncogene Proteins c-cbl; D010770:Phosphotransferases; D066246:ErbB Receptors; D008239:Lysine