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

We previously demonstrated that CR2 activation on human B lymphocyte surfac e triggered tyrosine phosphorylation of a p95 component and its interaction with p85 subunit of phosphatidylinositol 3' (PI 3) kinase, Despite identic al molecular mass of 95 kDa, this tyrosine phosphorylated p95 molecule was not CD19, the proto-oncogene Vav, or the adaptator Gab1, To identify this t yrosine phosphorylated p95 component, we first purified it by affinity chro matography on anti-phosphotyrosine mab covalently linked to Sepharose 4B, f ollowed by polyacrylamide gel electrophoresis. Then, the isolated 95-kDa ty rosine phosphorylated band was submitted to amino acid analysis by mass spe ctrometry; the two different isolated peptides were characterized by amino acid sequences 100% identical with two different domains of nucleolin, loca lized between aa 411-420 and 611-624, Anti-nucleolin mAb was used to confir m the antigenic properties of this p95 component. Functional studies demons trated that CR2 activation induced, within a brief span of 2 min, tyrosine phosphorylation of nucleolin and its interaction with Src homology 2 domain s 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 ident ical with those of the p95 previously described and induced by CR2 activati on. Furthermore, tyrosine phosphorylation of nucleolin was also induced in normal B lymphocytes by CR2 activation but neither by CD19 nor BCR activati on. These data support that tyrosine phosphorylation of nucleolin and its i nteraction with PI 3 kinase p85 subunit constitute one of the earlier steps in the specific intracellular signaling pathway of CR2.