Oksvold Morten P, Dagger Samantha A, Thien Christine B F, Langdon Wallace Y
School of Surgery and Pathology, University of Western Australia, Crawley, Western Australia 6009, Australia.
Mol Immunol. 2008 Feb;45(4):925-36. doi: 10.1016/j.molimm.2007.08.002. Epub 2007 Sep 14.
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.
指环型E3泛素连接酶Cbl-b在骨髓来源的肥大细胞（BMMC）中大量表达，并作为高亲和力IgE受体（FcεRI）信号反应的有效负调节因子发挥作用。为了确定Cbl-b E3连接酶活性的作用，我们构建了在指环结构域具有功能丧失突变的敲入小鼠。我们发现这些小鼠很健康，并且与等效的c-Cbl指环结构域突变小鼠不同，它们以预期频率产生纯合后代。对来自Cbl-b基因敲除小鼠和Cbl-b指环结构域突变小鼠的BMMC进行的比较分析表明，在所检测的大多数参数方面，与野生型细胞相比，二者的FcεRI信号传导均同样增强。一个显著的例外是，与指环结构域突变来源的细胞相比，Cbl-b基因敲除的BMMC中IκB激酶（IKK）的激活水平明显更高。此外，来自Cbl-b指环结构域突变小鼠的BMMC不会像基因敲除细胞那样明显延缓FcεRI的内化。然而，最引人注目的发现是，在FcεRI激活后，指环结构域突变的肥大细胞不会产生Cbl-b基因敲除培养物中明显可见的非常高水平的肿瘤坏死因子-α、白细胞介素-6和单核细胞趋化蛋白-1。因此，Cbl-b作为促进炎性细胞因子产生的FcεRI信号传导负调节因子的能力在很大程度上独立于指环结构域。
