The non-receptor tyrosine kinase Syk is a target of Cbl-mediated ubiquitylation upon B-cell receptor stimulation.
The negative regulator Cbl functions as a ubiquitin ligase towards activated receptor tyrosine kinases and facilitates their transport to lysosomes. Whether Cbl ubiquitin ligase activity mediates its negative regulatory effects on cytoplasmic tyrosine kinases of the Syk/ZAP-70 family has not been addressed, nor is it known whether these kinases are regulated via ubiquitylation during lymphocyte B-cell receptor engagement. Here we show that B-cell receptor stimulation in Ramos cells induces the ubiquitylation of Syk tyrosine kinase which is inhibited by a dominant-negative mutant of Cbl. Intact tyrosine kinase-binding and RING finger domains of Cbl were found to be essential for Syk ubiquitylation in 293T cells and for in vitro Syk ubiquitylation. These same domains were also essential for Cbl-mediated negative regulation of Syk as measured using an NFAT-luciferase reporter in a lymphoid cell. Association with Cbl did not alter the kinase activity of Syk. Altogether, our results support an essential role for Cbl ubiquitin ligase activity in the negative regulation of Syk, and establish that ubiquitylation provides a mechanism of Cbl-mediated negative regulation of cytoplasmic targets.
Rao
N
N
Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Ghosh
A K
AK
Ota
S
S
Zhou
P
P
Reddi
A L
AL
Hakezi
K
K
Druker
B K
BK
Wu
J
J
Band
H
H
D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009097:Multienzyme Complexes / Q000187:drug effects; D050722:Oncogene Protein v-cbl; D046988:Proteasome Endopeptidase Complex; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism*; D000072377:Syk Kinase; D025801:Ubiquitin / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D009097:Multienzyme Complexes; D050722:Oncogene Protein v-cbl; D011947:Receptors, Antigen, B-Cell; D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic; D025801:Ubiquitin; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex