Activation of Syk tyrosine kinase is required for c-Cbl-mediated ubiquitination of Fcepsilon RI and Syk in RBL cells.
Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells and basophils results in FcepsilonRI beta and gamma subunits ubiquitination by an as yet undefined mechanism. Here we show that, upon FcepsilonRI engagement on RBL-2H3 cells Syk undergoes ubiquitination and Syk kinase activity is required for its own ubiquitination and that of FcepsilonRI beta and gamma chains. This requirement was demonstrated by overexpression of Syk wild-type or its kinase-dead mutant in RBL cells or using an Syk-deficient RBL-derived cell line transfected with wild-type or a kinase inactive form of Syk. We also identify c-Cbl as the E3 ligase responsible for both Syk and receptor ubiquitination. Furthermore, we demonstrate that Syk controls tyrosine phosphorylation of Syk-associated Cbl induced after receptor engagement. These data suggest a mutual regulation between Syk and Cbl activities. Finally, we show that a selective inhibitor of proteasome degradation induces persistence of tyrosine-phosphorylated receptor complexes, of activated Syk, and of FcepsilonRI-triggered degranulation. Our results provide a molecular mechanism for down-regulation of engaged receptor complexes by targeting ubiquitinated FcepsilonRI and activated Syk to the proteasome for degradation.
Paolini
Rossella
R
Department of Experimental Medicine and Pathology, Institute Pasteur-Fondazione Cenci Bolognetti, University La Sapienza, Rome 00161, Italy. rosella.paolini@uniroma1.it
Molfetta
Rosa
R
Beitz
Laurie O
LO
Zhang
Juan
J
Scharenberg
Andrew M
AM
Piccoli
Mario
M
Frati
Luigi
L
Siraganian
Reuben
R
Santoni
Angela
A
D000818:Animals; D004792:Enzyme Precursors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D008024:Ligands; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D021122:Protein Subunits; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism*; D000072377:Syk Kinase; D019284:Thapsigargin / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014616:Vaccinia virus / Q000235:genetics
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D009842:Oligopeptides; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D017455:Receptors, IgE; D025801:Ubiquitin; D019284:Thapsigargin; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606898:Syk protein, rat; C067419:CBL protein, human; C078846:epoxomicin