Dual role of CbI links critical events in BCR endocytosis

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 CbI impacts BCR endocytosis and the underlying cytoskeletal dynamics. Specifically, we demonstrate that ligand-induced endocytosis is impaired in CbI-/- cells and that the ubiquitin ligase activity is required for CbI to promote endocytosis. We also show that phosphorylation of CrkII, activation of Rac downstream of CrkII and BCR capping require CbI. Furthermore, we show that the association of CbI and CrkII requires phosphorylation of CbI, but not its ubiquitin ligase activity. Our data indicate that CbI promotes BCR endocytosis and attenuates ligand-induced signaling by virtue of its ability to orchestrate receptor ubiquitylation and cytoskeletal dynamics.