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

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 (Fcvar epsilonRI). 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 Fcvar epsilonRI signalling compared to wild-type cells for most parameters examined. A notable exception was a markedly higher level of activation of IκB 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 Fcvar epsilonRI 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-α, IL-6, and MCP-1 evident in Cbl-b knockout cultures following Fcvar epsilonRI activation. Thus the ability of Cbl-b to function as a negative regulator of Fcvar epsilonRI signalling that promotes inflammatory cytokine production is largely independent of the RING finger domain.