Malek S N, Desiderio S
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1993 Oct 25;268(30):22557-65.
Several members of the Src family, including Blk, Lyn, Fyn(T), and Lck, are expressed in B cells. These kinases associate with the antigen receptor complex, and the activities of Blk, Fyn(T), and Lyn increase upon receptor engagement. Differences in the amino acid sequences and patterns of expression of these kinases suggest that they serve distinct functions. In this communication it is shown that the SH2 domains from Blk, Lyn, and Fyn(T) preferentially bind distinct sets of phosphoproteins from the mature B cell line A20. These interactions were found to depend on recognition of phosphotyrosine. The Blk SH2 domain bound more than 10 distinct phosphoprotein species, most of which reacted with an antiphosphotyrosine antibody; the phosphotyrosine content of these proteins was increased if surface immunoglobulin was cross-linked before extracts were made. Phosphoproteins of 72, 76, 115, and 130 kDa bound to the SH2 domains of Blk, Lyn, and Fyn(T). Phosphoamino acid analysis of these four proteins revealed that each contained phosphoserine, phosphothreonine, and phosphotyrosine. Proteins of 90 kDa, 130 kDa, and 150 kDa were preferentially bound by the Blk SH2 domain, while the Fyn(T) SH2 domain showed preferential binding to proteins of 76 and 180 kDa. The Lyn SH2 binding profile resembled that of Blk, but differences in the binding specificities of these kinases were also observed. Thus, among proteins that exhibit increased tyrosine phosphorylation following antigen receptor cross-linking, several have been identified that bind preferentially to SH2 domains of Blk, Fyn(T), or Lyn, suggesting that these kinases serve distinct functions. In addition, chimeric Fyn(T)-Blk SH2 domains were shown to be functional in binding assays and to exhibit binding specificities intermediate between those of the parent domains, consistent with the interpretation that the differences we observe in phosphoprotein binding by Fyn(T) and Blk SH2 domains reflect differences in their native structures.
Src家族的几个成员，包括Blk、Lyn、Fyn(T)和Lck，在B细胞中表达。这些激酶与抗原受体复合物相关联，并且在受体结合后，Blk、Fyn(T)和Lyn的活性会增加。这些激酶在氨基酸序列和表达模式上的差异表明它们具有不同的功能。在本通讯中表明，来自Blk、Lyn和Fyn(T)的SH2结构域优先结合成熟B细胞系A20中不同的磷酸化蛋白组。发现这些相互作用依赖于对磷酸酪氨酸的识别。Blk SH2结构域结合了10多种不同的磷酸化蛋白，其中大多数与抗磷酸酪氨酸抗体反应；如果在制备提取物之前对表面免疫球蛋白进行交联，则这些蛋白的磷酸酪氨酸含量会增加。72 kDa、76 kDa、115 kDa和130 kDa的磷酸化蛋白与Blk、Lyn和Fyn(T)的SH2结构域结合。对这四种蛋白的磷酸氨基酸分析表明，每种蛋白都含有磷酸丝氨酸、磷酸苏氨酸和磷酸酪氨酸。90 kDa、130 kDa和150 kDa的蛋白优先被Blk SH2结构域结合，而Fyn(T) SH2结构域则优先结合76 kDa和180 kDa的蛋白。Lyn SH2的结合谱与Blk相似，但也观察到这些激酶在结合特异性上的差异。因此，在抗原受体交联后酪氨酸磷酸化增加的蛋白中，已经鉴定出几种优先结合Blk、Fyn(T)或Lyn的SH2结构域的蛋白，这表明这些激酶具有不同的功能。此外，嵌合的Fyn(T)-Blk SH2结构域在结合试验中显示出功能，并表现出介于亲本结构域之间的结合特异性，这与我们观察到的Fyn(T)和Blk SH2结构域在磷酸化蛋白结合上的差异反映其天然结构差异的解释一致。
