Subcellular localization and signalling of Brutons tyrosine kinase (Btk)

Abstract: Brutons tyrosine kinase (Btk) is a non-receptor tyrosine kinase related to the Src family of kinases. Mutations in various parts of the gene have been shown to cause X-linked agammaglobulinemia (XLA), a primary immunodeficiency in humans, characterized by a defect in B-cell development. XLA patients lack B-cells and consequentially have very low levels of immunoglobulins in their serum. Thus, these patients suffer from an increased susceptibility mainly to extracellular bacterial infections. The molecular mechanism(s) underlying Btk localization, activation and signaling are not fully understood. We analyzed the subcellular localization of Btk employing a recombinant chimeric Btk fused with the Green Fluorescent Protein (GFP) with subsequent analysis of images using digital confocal microscopy. Different biochemical protein analyses were also performed. During this study we have found that Btk can translocate to the plasma membrane of living cells and play an important role as a potent inducer of cytoskeletal reorganization resulting in membrane ruffle formation. Moreover, we found that Btk can translocate to the nucleus and that Btk utilizes functional CRM-1 dependent nuclear export signal(s) to shuffle between the nucleus and the cytoplasm. We also found that Tec family kinases bind to caveolin-1, a major structural component of caveolae (rafts or microdomains) located in the plasma membrane. Finally, we demonstrate that Cbl acts as an E3-ubiquitin ligase for Btk and that ubiquitinated Btk is targeted for proteasomal degradation when Btk is expressed at high levels. Furthermore, upregulation of the small-ubiquitin-related-modifier (SUMO-1) downregulates Btk. In conclusion, the subcellular localization of Btk has implications regarding cytoskeletal regulation and /or potential targets inside the nucleus, which may be of relevance for B-cell development and differentiation. Also, Cbl-dependent ubiquitination as well as sumoylation are likely to provide a deeper insight into the negative regulation of Btk- mediated cell signaling.