Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase

Beston F Nore,Carl-Magnus Bäckesjö,Leonardo Vargas,C.I Edvard Smith,Abdalla J Mohamed,Birger Christensson

doi:10.1074/jbc.m006952200

Abstract

Bruton's tyrosine kinase (Btk), a nonreceptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases, has been shown to be critical for B cell proliferation, differentiation, and signaling. Loss-of-function mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA), a primary immunodeficiency in humans, and the less severe condition xid in mice. Although Btk is mainly localized in the cytoplasm under steady state conditions, it translocates to the plasma membrane upon growth factor stimulation and cross-linking of the B cell receptor. Nevertheless, in ectopically as well as endogenously Btk-expressing cells, it can also translocate to the nucleus. Deletion of the pleckstrin homology (PH) domain (DeltaPH1) leads, however, to an even redistribution of Btk within the nucleus and cytoplasm in the majority of transfected cells. In contrast, an SH3-deleted (DeltaSH3) mutant of Btk has been found to be predominantly nuclear. We also demonstrate that the nuclear accumulation of DeltaPH1 is dependent on Src expression. This nucleocytoplasmic shuttling is sensitive to the exportin 1/CRM1-inactivating drug, leptomycin B, indicating that Btk utilizes functional nuclear export signals. In addition, while the DeltaPH1 mutant of Btk was found to be active and tyrosine-phosphorylated in vivo, DeltaSH3 displayed decreased autokinase activity and was not phosphorylated. Our findings indicate that the nucleocytoplasmic shuttling of Btk has implications regarding potential targets inside the nucleus, which may be critical in gene regulation during B cell development and differentiation.

Highlights

Bruton’s tyrosine kinase (Btk), a nonreceptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases, has been shown to be critical for B cell proliferation, differentiation, and signaling
We have previously described a fusion construct consisting of Btk and green fluorescent protein (GFP), where the Btk cDNA is genetically hooked to the amino terminus of GFP (Btk-GFP) [13]
To obtain further insight into the mechanism of how Btk translocates to the nucleus of cells, we treated Btk-GFP-transfected COS-7 cells with the exportin 1 (CRM1)-inactivating antibiotic, leptomycin B

Summary

Introduction

Bruton’s tyrosine kinase (Btk), a nonreceptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases, has been shown to be critical for B cell proliferation, differentiation, and signaling. We demonstrate that the nuclear accumulation of ⌬PH1 is dependent on Src expression This nucleocytoplasmic shuttling is sensitive to the exportin 1/CRM1-inactivating drug, leptomycin B, indicating that Btk utilizes functional nuclear export signals. Btk has been shown to be predominantly cytoplasmic and can translocate to the plasma membrane upon, for example, growth factor stimulation of cells, a small pool of the protein can translocate to the nucleus under certain physiological growth conditions [13]. Proteins that have functions in both the nucleus and cytoplasm contain nuclear export signals (NES) in addition to the NLS. We provide evidence that Btk utilizes functional CRM1-dependent nuclear export signal(s) to shuttle between the nucleus and the cytoplasm. In Src-deficient fibroblasts, the nucleocytoplasmic shuttling of a deleted PH domain mutant of Btk was found to be dependent on c-Src expression

Methods

Results

Conclusion