The Cbl-b RING finger domain has a limited role in regulating inflammatory cytokine production by IgE-activated mast cells

Abstract

The RING finger type E3 ubiquitin ligase, Cbl-b, is abundantly expressed in bone marrow-derived mast cells (BMMCs) and functions as a potent negative regulator of signalling responses from the high-affinity IgE receptor (FcvarepsilonRI). To determine the contribution of Cbl-b E3 ligase activity we generated knockin mice with a loss-of-function mutation in the RING finger domain. We find the mice to be healthy and, unlike equivalent c-Cbl RING finger mutant mice, produce homozygous offspring at the expected frequency. Comparative analyses of BMMCs from Cbl-b knockout and Cbl-b RING finger mutant mice revealed that both showed similarly enhanced FcvarepsilonRI signalling compared to wild-type cells for most parameters examined. A notable exception was a markedly higher level of activation of IkappaB kinase (IKK) in Cbl-b knockout BMMC compared to RING finger mutant-derived cells. In addition BMMCs from the Cbl-b RING finger mutant did not retard FcvarepsilonRI internalization to the extent observed for knockout cells. Most striking however was the finding that RING finger mutant mast cells do not produce the very high levels of TNF-alpha, IL-6, and MCP-1 evident in Cbl-b knockout cultures following FcvarepsilonRI activation. Thus the ability of Cbl-b to function as a negative regulator of FcvarepsilonRI signalling that promotes inflammatory cytokine production is largely independent of the RING finger domain.

Mechanisms of NKT cell anergy induction involve Cbl-b-promoted monoubiquitination of CARMA1

“…To understand the importance of the Cbl-b E3 ligase activity, we analyzed the Cbl-b knockin mice containing a mutation in the critical cysteine 373 of the RING finger domain to alanine (C373A) (13). Cbl-b C373A mutant knockin mice did not change the NKT cell populations in the spleen (Fig.…”

Essential Role of E3 Ubiquitin Ligase Activity in Cbl-b–Regulated T Cell Functions

“…In contrast, Cbl-b ligase activity has been reported to be differentially required for IgE receptor-mediated mast cell functions. Whereas loss of Cbl-b enzymatic activity altered some signaling pathways downstream of the FcεRI receptor, Cbl-b negative regulation of the signaling pathway leading to the production of inflammatory cytokines was found to be largely independent of the RING finger domain (30). Thus, Cbl-b may biochemically function as both E3 ligase and multivalent adapter protein depending on the cellular context or even in different signaling pathways within each cell type.…”

“…) knock-in mice were generated by homologous recombination as previously described (30). In brief, a targeting vector was constructed to introduce a cysteine (TGC) to alanine (GCG) substitution at amino acid position 373 encoded within the RING finger domain (at exon 8) of Cbl-b.…”

“…To dissect genetically the enzymatic function of Cbl-b in vivo, we analyzed mice that carry a RING finger inactivating point mutation (C373A KI/KI ) (30). These mice are viable, fertile, and grossly normal.…”

Indispensable roles of mammalian Cbl family proteins as negative regulators of protein tyrosine kinase signaling

“…So far, such "knock-in" mutants have been reported for the Cbl TKB (G304E, equivalent to G306E in human Cbl), 29 and RING finger (C379A, equivalent to C381A in human Cbl), 30 domains, as well as one of the C-terminal region tyrosine residues (Y737F, equivalent to Y731F in human Cbl), 31 that upon phosphorylation is known to interact with p85 subunit of PI3K, as well as for the Cbl-b RING finger domain (C373A, equivalent to C373A in human Cbl-b). 32 Knock-in mutants highlight the importance of specific interactions and also demonstrate the complexity of in vivo analyses based on in vitro biochemistry. For instance, the TKB domain mutation was originally identified in C. elegans as a loss-of-function mutation.…”