Jacob Michele, Todd Leslie, Sampson Maame F, Puré Ellen
Wistar Institute and Ludwig Institute for Cancer Research, 3601 Spruce Street, Philadelphia, PA 19104-4268, USA.
Int Immunol. 2008 Apr;20(4):485-97. doi: 10.1093/intimm/dxn010. Epub 2008 Feb 17.
Receptor endocytosis down-regulates ligand-induced signaling in a timely manner and depends on cytoskeletal remodeling. In B lymphocytes, internalization of B cell receptors (BCRs) is also critical to antigen presentation. However, the mechanisms underlying BCR endocytosis are not fully understood. Similarly, the molecular mechanisms linking endocytosis to cytoskeletal remodeling remain poorly defined. We used flow cytometry, pull-down assays, immunochemistry and fluorescence microscopy to investigate BCR internalization in the DT40 B cell line. We demonstrate that ablation of Cbl impacts BCR endocytosis and the underlying cytoskeletal dynamics. Specifically, we demonstrate that ligand-induced endocytosis is impaired in Cbl-/- cells and that the ubiquitin ligase activity is required for Cbl to promote endocytosis. We also show that phosphorylation of CrkII, activation of Rac downstream of CrkII and BCR capping require Cbl. Furthermore, we show that the association of Cbl and CrkII requires phosphorylation of Cbl, but not its ubiquitin ligase activity. Our data indicate that Cbl promotes BCR endocytosis and attenuates ligand-induced signaling by virtue of its ability to orchestrate receptor ubiquitylation and cytoskeletal dynamics.
受体胞吞作用能及时下调配体诱导的信号传导，且依赖于细胞骨架重塑。在B淋巴细胞中，B细胞受体（BCR）的内化作用对抗抗原呈递也至关重要。然而，BCR胞吞作用的潜在机制尚未完全明确。同样，将胞吞作用与细胞骨架重塑联系起来的分子机制也仍不清楚。我们使用流式细胞术、下拉分析、免疫化学和荧光显微镜来研究DT40 B细胞系中的BCR内化作用。我们证明Cbl的缺失会影响BCR胞吞作用以及潜在的细胞骨架动力学。具体而言，我们证明在Cbl基因敲除细胞中，配体诱导的胞吞作用受损，且Cbl促进胞吞作用需要泛素连接酶活性。我们还表明，CrkII的磷酸化、CrkII下游Rac的激活以及BCR封帽都需要Cbl。此外，我们表明Cbl与CrkII的结合需要Cbl的磷酸化，但不需要其泛素连接酶活性。我们的数据表明，Cbl凭借其协调受体泛素化和细胞骨架动力学的能力促进BCR胞吞作用并减弱配体诱导的信号传导。
