Abstract
A fraction of Bruton's tyrosine kinase (Btk) co-localizes with actin fibers upon stimulation of mast cells via the high affinity IgE receptor (FcepsilonRI). In this study, a molecular basis of the Btk co-localization with actin fibers is presented. Btk and other Tec family tyrosine kinases have a pleckstrin homology (PH) domain at their N termini. The PH domain is a short peptide module frequently found in signal-transducing proteins and cytoskeletal proteins. Filamentous actin (F-actin) is shown to be a novel ligand for a subset of PH domains, including that of Btk. The actin-binding site was mapped to a 10-residue region of the N-terminal region of Btk. Basic residues in this short stretch are demonstrated to be involved in actin binding. Isolated PH domains induced actin filament bundle formation. Consistent with these observations, Btk binds F-actin in vitro and in vivo. Wild-type Btk protein is in part translocated to the cytoskeleton upon FcepsilonRI cross-linking, whereas Btk containing a mutated PH domain is not. Phosphatidylinositol 3,4, 5-trisphosphate-mediated membrane translocation of Btk was enhanced in cytochalasin D-pretreated, FcepsilonRI-stimulated mast cells. These data indicate that PH domain-mediated F-actin binding plays a role in Btk co-localization with actin filaments.
Highlights
Bruton’s tyrosine kinase (Btk),1 a cytoplasmic protein-tyrosine kinase (PTK), is implicated in signal transduction initiated by numerous immune cell receptors including Fc⑀RI and B cell antigen receptor
Btk Is Co-localized with the Actin Fibers upon Fc⑀RI Crosslinking—Fc⑀RI cross-linking induces dramatic cytoskeletal changes accompanied by increased form of actin (F-actin) content, membrane ruffling, increased cell adhesion and spreading, and the formation of actin-rich adhesion structures, termed actin plaques [44]
Upon Fc⑀RI cross-linking, co-staining of F-actin with rhodamine-phalloidin showed that a portion of the Btk protein pool is co-localized with F-actin in a spacio-temporally specific manner
Summary
A cytoplasmic protein-tyrosine kinase (PTK), is implicated in signal transduction initiated by numerous immune cell receptors including Fc⑀RI and B cell antigen receptor (reviewed in Refs. 1 and 2). Upon B cell receptor stimulation, Btk is recruited to the plasma membrane through the interaction between the PH domain and phosphatidylinositol 3,4,5-trisphosphate (PIP3) [5,6,7] It is activated by phosphorylation at the activation loop (Tyr-551) by Lyn or Syk [8, 9]. Several studies [26, 30, 31] showed that various PH domains bind to phosphatidylinositol 4,5bisphosphate (PIP2) and related molecules through the positively charged residues in their N-terminal halves. Our previous studies [34, 35] demonstrated that multiple isoforms of protein kinase C (PKC) interact with several PH domains, including that of Btk. Btk was shown to be phosphorylated and enzymatically down-regulated by PKC in vitro and in vivo. Consistent with these in vitro data, a fraction of Btk was translocated from the cytosol to the cytoskeleton to co-localize with actin fibers upon Fc⑀RI cross-linking
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