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 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.
Barel
M
M
Immunochimie des Régulations Cellulaires et des Interactions Virales, Centre Institut National de la Santé et de la Recherche Médicale, Paris, France.
Le Romancer
M
M
Frade
R
R
D018941:Antigens, CD19 / Q000378:metabolism; D001402:B-Lymphocytes / Q000201:enzymology / Q000378:metabolism* / Q000821:virology; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D020014:K562 Cells; D016393:Lymphoma, B-Cell / Q000201:enzymology / Q000276:immunology / Q000378:metabolism / Q000821:virology; D008565:Membrane Proteins / Q000378:metabolism* / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D000090063:Proto-Oncogene Mas; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D017464:Receptors, Complement 3d / Q000378:metabolism* / Q000502:physiology; D014407:Tumor Cells, Cultured; D000097796:Nucleolin
D018941:Antigens, CD19; D008565:Membrane Proteins; D010446:Peptide Fragments; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D019000:Phosphotyrosine; D000090063:Proto-Oncogene Mas; D016601:RNA-Binding Proteins; D011947:Receptors, Antigen, B-Cell; D017464:Receptors, Complement 3d; D000097796:Nucleolin; C000715470:MAS1 protein, human