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.
