CIN85/CD2AP-based protein complexes in B cell antigen receptor signalling

Abstract

Early B cell antigen receptor (BCR) signal transduction involves formation of multimeric protein complexes and their recruitment to BCR-containing areas in the plasma membrane. One such membrane-targeted signalosome is the Ca2+ initiation complex that is assembled on the central adaptor protein SH2 domain-containing leukocyte protein of 65 kDa (SLP65). To decipher the mechanism of SLP65 membrane localisation, the interactome of SLP65 was analysed by our group and identified the scaffolds Cbl-interacting protein of 85 kDa (CIN85) and CD2-associated protein (CD2AP) to be constantly associated with SLP65. Further studies showed that disruption of this interaction by changes of SLP65 binding motifs resulted in impaired BCR-induced Ca2+ mobilisation in DT40 and primary murine B cells. Hence, the following questions arose for this thesis: Does ablation of CIN85 and CD2AP result in a BCR-induced Ca2+ mobilisation defect as shown on the SLP65 side and, thus, do CIN85, CD2AP and SLP65 comprise a preformed signal transducer complex? And, how do CIN85 and CD2AP then contribute to SLP65 function in BCR signal transduction? In my thesis, I demonstrated that CIN85 positively regulated the rapid onset and strength of BCR-induced Ca2+ mobilisation in DT40 B cells. However, CD2AP could partially replace CIN85 function in this respect. Using live cell imaging I showed that CIN85 and CD2AP translocate to the plasma membrane upon stimulation of the BCR, but used different anchoring modes. Interestingly, the very same domains of CIN85 necessary for BCRinduced Ca2+ signalling provided CIN85 with access to the plasma membrane. More detailed analysis of membrane localisation using total internal reflection microscopy identified CIN85 to colocalise with BCR-containing microclusters upon BCR engagement. In contrast to CIN85 only very few CD2AP molecules were found in these microclusters. Importantly, the preformation of CIN85 with SLP65 could be bypassed by providing SLP65 with direct access to the BCR. This implies that CIN85 and SLP65 comprise a preformed BCR transducer module. In this complex CIN85 targets SLP65 directly to BCR-containing microclusters thereby enabling efficient BCR-induced Ca2+ responses. Collectively, the herein documented results contribute to the understanding of BCR activation with respect to BCR-proximal signalosomes and their importance in the transduction of BCR signals.