Immunoaffinity isolation of Btk´s signalosome, a proteomic approach to identifying interacting proteins

Abstract: The Signalosome is a term used to define a putative signalling complex, which assembles near the plasma membrane in response to external signals received at cell surface receptors and then migrates towards downstream effectors. It is proposed to regulate the level of intracellular Ca2+ and subsequent downstream signalling events. To date it has been defined to consist of BTK, BLNK, BCAP, VAV, PLCγ2 and PI3K1-4 in B-Cells. This work entailed initiating a new proteomic approach to investigate the nature and extent of Bruton’s tyrosine kinase, Btk, involvement in the signalosome – inherently, the aim was to study multiple interactions of Btk with other molecules. By transfecting host cells with a Btk gene-transfer plasmid, virus particles were produced that were used to up-regulate and analyse the expression of Btk in three haematopoietic cell lines: B-cells, Pre-B-cells and a myeloid cancer cell. The construction of a new gene-transfer vector was successfully carried out by plasmid sub-cloning and it was subsequently found to effectively transfect the host cells and produce virus particles. The recombinant virus particles were employed with success in transducing three haematopoietic cell lines and with immunopurification and subsequent gel separation protein signalosome complexes were obtained ready for analysis by mass spectrometrical fingerprinting (to be carried out as a joint effort in Mount Sinai Hospital in Toronto, Canada).