Baba Y, Nonoyama S, Matsushita M, Yamadori T, Hashimoto S, Imai K, Arai S, Kunikata T, Kurimoto M, Kurosaki T, Ochs H D, Yata J i, Kishimoto T, Tsukada S
Department of Medicine III, Osaka University Medical School, Osaka, Japan.
Blood. 1999 Mar 15;93(6):2003-12.
Bruton's tyrosine kinase (Btk) has been shown to play a role in normal B-lymphocyte development. Defective expression of Btk leads to human and murine immunodeficiencies. However, the exact role of Btk in the cytoplasmic signal transduction in B cells is still unclear. This study represents a search for the substrate for Btk in vivo. We identified one of the major phosphoproteins associated with Btk in the preB cell line NALM6 as the Wiskott-Aldrich syndrome protein (WASP), the gene product responsible for Wiskott-Aldrich syndrome, which is another hereditary immunodeficiency with distinct abnormalities in hematopoietic cells. We demonstrated that WASP was transiently tyrosine-phosphorylated after B-cell antigen receptor cross-linking on B cells, suggesting that WASP is located downstream of cytoplasmic tyrosine kinases. An in vivo reconstitution system demonstrated that WASP is physically associated with Btk and can serve as the substrate for Btk. A protein binding assay suggested that the tyrosine-phosphorylation of WASP alters the association between WASP and a cellular protein. Furthermore, identification of the phosphorylation site of WASP in reconstituted cells allowed us to evaluate the catalytic specificity of Btk, the exact nature of which is still unknown.
布鲁顿酪氨酸激酶（Btk）已被证明在正常B淋巴细胞发育中起作用。Btk表达缺陷会导致人类和小鼠免疫缺陷。然而，Btk在B细胞胞质信号转导中的确切作用仍不清楚。本研究旨在寻找Btk在体内的底物。我们在preB细胞系NALM6中鉴定出与Btk相关的一种主要磷蛋白为威斯科特-奥尔德里奇综合征蛋白（WASP），该基因产物导致威斯科特-奥尔德里奇综合征，这是另一种遗传性免疫缺陷，在造血细胞中有明显异常。我们证明，在B细胞上进行B细胞抗原受体交联后，WASP会短暂地发生酪氨酸磷酸化，这表明WASP位于胞质酪氨酸激酶的下游。体内重建系统表明，WASP与Btk存在物理关联，并且可以作为Btk的底物。蛋白质结合试验表明，WASP的酪氨酸磷酸化改变了WASP与一种细胞蛋白之间的关联。此外，对重建细胞中WASP磷酸化位点的鉴定使我们能够评估Btk的催化特异性，其确切性质仍然未知。
