Activation of the EBV/C3d receptor (CR2, CD21) on human B lymphocyte surface triggers tyrosine phosphorylation of the 95-kDa nucleolin and its interaction with phosphatidylinositol 3 kinase
Activation of the EBV/C3d receptor (CR2, CD21) on human B lymphocyte surface triggers tyrosine phosphorylation of the 95-kDa nucleolin and its interaction with phosphatidylinositol 3 kinase
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DOI：
10.4049/jimmunol.166.5.3167
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发表时间：
2001-03-01
影响因子：
4.4
通讯作者：
Frade, R
中科院分区：
文献类型：
--
作者：
Barel, M;Le Romancer, M;Frade, R
我们先前已证明，人B淋巴细胞表面的CR2活化会触发一种p95成分的酪氨酸磷酸化及其与磷脂酰肌醇3'（PI 3）激酶的p85亚基的相互作用。尽管分子量均为95kDa，但这种酪氨酸磷酸化的p95分子不是CD19、原癌基因Vav或衔接蛋白Gab1。为了鉴定这种酪氨酸磷酸化的p95成分，我们首先通过与琼脂糖4B共价连接的抗磷酸酪氨酸单克隆抗体亲和层析对其进行纯化，随后进行聚丙烯酰胺凝胶电泳。然后，将分离出的95 - kDa酪氨酸磷酸化条带通过质谱进行氨基酸分析；两种不同的分离肽的氨基酸序列特征与核仁素的两个不同结构域100%相同，分别位于氨基酸411 - 420和611 - 624之间。使用抗核仁素单克隆抗体来确认这种p95成分的抗原特性。功能研究表明，CR2活化在短短2分钟内诱导核仁素的酪氨酸磷酸化及其与PI 3激酶的p85亚基以及3BP2和Grb2的Src同源2结构域的相互作用，但不与Fyn和Gap的Src同源2结构域相互作用。核仁素的这些特性与先前描述的由CR2活化诱导的p95的特性相同。此外，在正常B淋巴细胞中，核仁素的酪氨酸磷酸化也由CR2活化诱导，而不由CD19或BCR活化诱导。这些数据支持核仁素的酪氨酸磷酸化及其与PI 3激酶p85亚基的相互作用构成了CR2特异性细胞内信号通路中的早期步骤之一。
We previously demonstrated that CR2 activation on human B lymphocyte surface triggered tyrosine phosphorylation of a p95 component and its interaction with p85 subunit of phosphatidylinositol 3' (PI 3) kinase, Despite identical molecular mass of 95 kDa, this tyrosine phosphorylated p95 molecule was not CD19, the proto-oncogene Vav, or the adaptator Gab1, To identify this tyrosine phosphorylated p95 component, we first purified it by affinity chromatography on anti-phosphotyrosine mab covalently linked to Sepharose 4B, followed by polyacrylamide gel electrophoresis. Then, the isolated 95-kDa tyrosine phosphorylated band was submitted to amino acid analysis by mass spectrometry; the two different isolated peptides were characterized by amino acid sequences 100% identical with two different domains of nucleolin, localized between aa 411-420 and 611-624, Anti-nucleolin mAb was used to confirm the antigenic properties of this p95 component. Functional studies demonstrated that CR2 activation induced, within a brief span of 2 min, tyrosine phosphorylation of nucleolin and its interaction with Src homology 2 domains of the p85 subunit of PI 3 kinase and of 3BP2 and Grb2, but not with Src homology 2 domains of Fyn and Gap. These properties of nucleolin were identical with those of the p95 previously described and induced by CR2 activation. Furthermore, tyrosine phosphorylation of nucleolin was also induced in normal B lymphocytes by CR2 activation but neither by CD19 nor BCR activation. These data support that tyrosine phosphorylation of nucleolin and its interaction with PI 3 kinase p85 subunit constitute one of the earlier steps in the specific intracellular signaling pathway of CR2.