Rao N, Ghosh A K, Ota S, Zhou P, Reddi A L, Hakezi K, Druker B K, Wu J, Band H
Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
EMBO J. 2001 Dec 17;20(24):7085-95. doi: 10.1093/emboj/20.24.7085.
The negative regulator Cbl functions as a ubiquitin ligase towards activated receptor tyrosine kinases and facilitates their transport to lysosomes. Whether Cbl ubiquitin ligase activity mediates its negative regulatory effects on cytoplasmic tyrosine kinases of the Syk/ZAP-70 family has not been addressed, nor is it known whether these kinases are regulated via ubiquitylation during lymphocyte B-cell receptor engagement. Here we show that B-cell receptor stimulation in Ramos cells induces the ubiquitylation of Syk tyrosine kinase which is inhibited by a dominant-negative mutant of Cbl. Intact tyrosine kinase-binding and RING finger domains of Cbl were found to be essential for Syk ubiquitylation in 293T cells and for in vitro Syk ubiquitylation. These same domains were also essential for Cbl-mediated negative regulation of Syk as measured using an NFAT-luciferase reporter in a lymphoid cell. Association with Cbl did not alter the kinase activity of Syk. Altogether, our results support an essential role for Cbl ubiquitin ligase activity in the negative regulation of Syk, and establish that ubiquitylation provides a mechanism of Cbl-mediated negative regulation of cytoplasmic targets.
负调节因子Cbl作为一种泛素连接酶作用于活化的受体酪氨酸激酶，并促进其向溶酶体的转运。Cbl泛素连接酶活性是否介导其对Syk/ZAP-70家族细胞质酪氨酸激酶的负调节作用尚未得到研究，在淋巴细胞B细胞受体参与过程中这些激酶是否通过泛素化进行调节也不清楚。在此我们表明，Ramos细胞中的B细胞受体刺激诱导Syk酪氨酸激酶的泛素化，而这种泛素化被Cbl的显性负性突变体所抑制。发现Cbl完整的酪氨酸激酶结合域和RING指结构域对于293T细胞中的Syk泛素化以及体外Syk泛素化至关重要。在淋巴样细胞中使用NFAT荧光素酶报告基因检测时，这些相同的结构域对于Cbl介导的Syk负调节也至关重要。与Cbl的结合并未改变Syk的激酶活性。总之，我们的结果支持Cbl泛素连接酶活性在Syk负调节中起重要作用，并证实泛素化提供了一种Cbl介导的对细胞质靶点负调节的机制。
