The non-receptor tyrosine kinase Syk is a target of Cbl-mediated ubiquitylation upon B-cell receptor stimulation

Abstract

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 ®nger 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.

The Shb signalling scaffold binds to and regulates constitutive signals from the Epstein–Barr virus LMP2A membrane protein

“…LV Matskova et al Shb and AIP4 regulate Syk stability in LMP2A positive but not in control cells Downregulation of the Syk tyrosine kinase in normal cells has been attributed to ubiquitination of the activated kinase by the Cbl RING-domain E3 ubiquitin ligase (Ota et al, 2000;Rao et al, 2001). Results from our group (Winberg et al, 2000) and others (Ikeda et al, 2000) show that AIP4 and Nedd4-like HECT-domain E3-ligases are recruited by LMP2A and subsequently catalyse ubiquitination of Syk.…”

Src-like adaptor protein (SLAP) regulates B cell receptor levels in a c-Cbl-dependent manner

“…Second, SLAP may function to adapt the E3 ubiquitin ligase, c-Cbl, to the BCR complex, facilitating the ubiquitination of components of the BCR complex. A number of signaling proteins downstream of the BCR previously have been shown to be ubiquitinated by c-Cbl, including Lyn and Syk (17,26,27). Previous studies have not addressed whether other components of the BCR complex are ubiquitinated.…”

Cloning and characterization of human Src-like adaptor protein 2 and a novel splice isoform, SLAP-2-v

“…The mechanism by which c-Cbl negatively regulates SYK and ZAP-70 is not fully understood, however, it has been proposed that c-Cbl ubiquitin ligase activity may be involved in this process. Recently, SYK has been shown to be a direct target for c-Cbl ubiquitination in Bcells (Rao et al, 2001), and through an adaptor function of ZAP-70, c-Cbl has been demonstrated to ubiquitinate the TCR zeta chain, a critical component of the TCR : CD3 complex (Wang et al, 2001). c-Cbl-mediated ubiquitination of components of the TCR could result in either degradation via the proteasome or, alternatively, could serve as a signal for trafficking of the activated TCR complex to the lysosome.…”