Regulation of ubiquitin protein ligase activity in c-Cbl by phosphorylation-induced conformational change and constitutive activation by tyrosine to glutamate point mutations.
c-Cbl down-regulates receptor tyrosine kinases by conjugating ubiquitin to them, leading to receptor internalization and degradation. The ubiquitin protein ligase activity of c-Cbl (abbreviated as E3 activity) is mediated by its RING finger domain. We show here that the E3 activity of c-Cbl is negatively regulated by other domains present in the amino-terminal half of the protein (the TKB and linker helix domains) and that this negative regulation is removed when the protein is phosphorylated on tyrosine residues. Protease digestion studies indicate that tyrosine phosphorylation alters the conformation of c-Cbl. We also show that mutation of certain conserved tyrosine residues to glutamate can constitutively activate the E3 activity of c-Cbl. In particular, a Y371E mutant shows constitutive E3 activity while retaining the ability to bind epidermal growth factor receptor (EGFR). The Y371E mutant also has altered protease sensitivity from wild type, instead resembling the proteolytic pattern seen with tyrosine-phosphorylated c-Cbl. Mutation of the homologous tyrosine residue in Cbl-b to glutamate also leads to E3 activation while retaining EGFR-binding ability. These studies argue that Tyr-371 plays a key role in activating the E3 activity of c-Cbl and that the Y371E mutant may partially mimic phosphorylation at that site. However, Tyr-371 point mutants of c-Cbl are still able to undergo phosphorylation-induced E3 activation, and we show that Tyr-368 can also be phosphorylated in addition to Tyr-371, and contributes to activation.
Kassenbrock
C Kenneth
CK
Department of Pathology, University of Colorado Health Sciences Center, 4200 E Ninth Avenue, Denver, CO 80262, USA.
Anderson
Steven M
SM
D002460:Cell Line; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018698:Glutamic Acid / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D014443:Tyrosine / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis* / Q000378:metabolism*
D011518:Proto-Oncogene Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D066246:ErbB Receptors; C067419:CBL protein, human