CIN85 in proximal and distant B cell antigen receptor signaling
von Kathrin Schulz
Datum der mündl. Prüfung:2016-02-29
Betreuer:Prof. Dr. Jürgen Wienands
Gutachter:Prof. Dr. Jürgen Wienands
Gutachter:Prof. Dr. Henning Urlaub
EnglischB cell antigen receptor (BCR) signaling is pivotal in B cell development and initiation of humoral immune responses. In this process, numerous proteins are involved that interact with each other and build up a sophisticated interplay of activating as well as terminating complexes to generate an appropriate signal outcome. Recently, two boys were identified who showed deficiencies for serum immunoglobulins leading to recurrent infections due to a big deletion in the gene coding for the adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). CIN85 is known to be a constitutive interactor of the adaptor protein SLP65 (SH2 domain-containing leukocyte protein of 65 kDa) and supports the plasma membrane recruitment of SLP65 as a platform for the assembly of the Ca2+ initiation complex in the chicken DT40 B cell line. Furthermore, involvement of CIN85 is reported in the activation of the BCR-induced canonical NF-κB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) pathway in primary mouse B cells. However, the precise mechanism of CIN85 plasma membrane recruitment and hence provided support for SLP65 function as well as NF-κB activation is unknown. Here I show that CIN85 regulates plasma membrane recruitment of SLP65 and the known canonical NF-κB pathway activator PKCβ (Protein kinase C beta) in the human DG75 B cell line. Based on the generation of a human CIN85-deficient B cell sub-line, I found that CIN85 supports BCR-induced SLP65 membrane recruitment probably due to direct and/or ubiquitin mediated interaction with proteins present in BCR signalosomes as well as by direct association with the short cytoplasmic tail of the IgM-BCR. With confocal microscopy, I could provide evidence for the importance of CIN85 in respect to PKCβ localization and hence narrow the point of CIN85 interaction in enhancing BCR-induced NF-κB signaling. Moreover, I could show for the first time that CIN85 acts as a stabilizer of the direct SH3 domain interacting protein SLP65. Collectively, my results demonstrate that CIN85 exerts diverse roles in different stages of BCR-induced signaling and hence highlight the importance of this adaptor protein. Thus, the results of my PhD project contribute to a better understanding of the complex BCR signaling cascade. Noteworthy, the performance of genetic and biochemical experiments in the generated model systems provide further insights into the mechanistic basis of the patients phenotype and at the same time open new opportunities for future investigations.
Keywords: CIN85; SLP65; NF-kappaB; BCR signaling