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

Abstract

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.

Up-regulation of the KLF2 Transcription Factor by Fluid Shear Stress Requires Nucleolin

“…4A). Importantly, the sizes of the catalytic nucleolin forms noted in our experiments correspond to previous reports of multiple nucleolin forms (33)(34)(35)(36)(37). The formation of these catalytic forms was blocked when the cells were pretreated with the PI3K inhibitor LY294002.…”

“…The exact size and predominance of a given species depends both on cell type and stimulus, but the smaller forms do exhibit independent functional specificity (35, 44) (e.g. the 95-kDa species of nucleolin that has been found to interact with the p85 regulatory subunit of PI3K) (34). The production of these forms probably results from the high susceptibility of nucleolin to post-translational regulation (48).…”

“…Nucleolin Interacts with PI3K and hnRNP-D-Nucleolin has been reported to directly bind the p85 regulatory subunit of PI3K upon activation of the CR2 cell surface receptor on human B lymphocytes (34). Considering the dependence of nucleolin binding upon PI3K activity in endothelial cells, we sought to determine if a nucleolin-PI3K interaction was occurring in our experiments.…”

Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells

“…The nucleolin/PI-3K interaction has been reported to be induced in other two different cellular systems described in the literature. In normal B lymphocytes, CD21 activation induced tyrosine phosphorylation of nucleolin which interacted with Scr homology 2 (SH2) domain of p85 subunit of PI3-K [51]. In endothelial cells, the application of fluid stress also induced a nucleolin/PI-3K interaction [52] although the mechanism of direct tyrosine phosphorylation of nucleolin by fluid stress was not determined.…”

AU-rich RNA binding proteins in hematopoiesis and leukemogenesis

“…67 In response to CD21 activation, it also becomes tyrosine phosphorylated and associates with the unphosphorylated p85 subunit of the PI3K pathway. 68 Finally, through RNA-dependent association with DGCR8, nucleolin may serve an adaptor function in transferring the pri-miRNAs to the nucleolus and in the processing-cleavage of pri-miRNAs to pre-miRNA within the nucleolus by a Drosha-DGCR8-nucleolin complex. 69 As summarized in Table 1, deregulated expression of nucleolin is a consistent feature of several types of leukemia.…”