c-Cbl binding and ubiquitin-dependent lysosomal degradation of membrane-associated Notch1.
Regulation of Notch1 activity is critical for cell fate decisions and differentiation of skeletal myoblasts. We have employed the skeletal myoblast cell line C2C12 to study posttranslational regulation of Notch1 protein levels during myogenesis. Although the major degradation pathway of the activated intracellular Notch1 fragment appears to involve ubiquitination and degradation by the 26 S proteasome, we provide evidence for an alternative catalytic pathway where the endogenous, transmembrane form of Notch1 is targeted to the lysosomal compartment. Immunoprecipitation analysis revealed ubiquitin-dependent accumulation of transmembrane Notch1 protein after treatment with the lysosomal inhibitor chloroquine but not after treatment with various proteasome inhibitors. This finding was supported by the observation that the transmembrane form of Notch1 was tyrosine-phosphorylated and specifically coprecipitated with the ubiquitin ligase c-Cbl. Our data suggest a regulatory mechanism down-regulating Notch1 protein levels already at the cellular surface, possibly with consequences for Notch-dependent signal transduction during terminal differentiation processes.
Jehn
Birgit M
BM
Friedrich-Alexander University, Erlangen/Nuremberg, Department of Experimental Medicine I, 91054 Erlangen, Germany.
Dittert
Irina
I
Beyer
Stephanie
S
von der Mark
Klaus
K
Bielke
Wolfgang
W
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D012333:RNA, Messenger / Q000378:metabolism; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D008565:Membrane Proteins; C497762:Notch1 protein, mouse; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D014157:Transcription Factors; D025801:Ubiquitin; D014443:Tyrosine; D002738:Chloroquine; D004492:Edetic Acid; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C493849:Cbl protein, mouse