Enhancement of B‐cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism

Abstract

Tyrosine phosphorylation of adaptor protein c‐Abl‐Src homology 3 (SH3) domain‐binding protein‐2 (3BP2, also referred to SH3BP2) positively regulates the B‐cell antigen receptor (BCR)‐mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B‐cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2‐P416R, corresponding to P418R in human protein) enhanced BCR‐mediated activation of NFAT. 3BP2‐P416R increased the signaling complex formation with Syk, phospholipase C‐γ2 (PLC‐γ2), and Vav1. In contrast, 3BP2‐P416R could not change the association with the negative regulator 14‐3‐3. Loss of the association mutant that was incapable to associate with 14‐3‐3 could not mimic BCR‐mediated NFAT activation in Syk‐deficient cells. Moreover, BCR‐mediated phosphorylation of extracellular signal regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules.

Dectin-1-mediated Signaling Leads to Characteristic Gene Expressions and Cytokine Secretion via Spleen Tyrosine Kinase (Syk) in Rat Mast Cells

“…After the stimulation, cells were washed with ice-cold PBS twice and solubilized in the lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 100 mM NaF, 1 mM Na 3 VO 4 , 1 mM PMSF, and 2 g/ml aprotinin) containing 1% Triton X-100. Preparation of detergentsoluble cell lysates, immunoprecipitation, and immunoblotting were performed as described previously (32)(33)(34). Pull-down Assay-The GST-rat Syk-SH2 (both N-and C-terminal SH2 domains) expression construct was a gift from Dr. Reuben P. Siraganian.…”

“…5 ϫ 10 6 cells were incubated without or with 100 g/ml curdlan in medium and were solubilized with the lysis buffer containing 1% Triton X-100. In vitro binding experiments were performed as described previously (32)(33)(34).…”

Association of C-Type Lectin Mincle with FcεRIβγ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk

“…In some experiments, Syk inhibitors (R406 and BAY61-3606) were added to the culture medium before stimulation. Preparation of cell lysates and immunoprecipitation were performed as described previously165657585960.…”

“…Proteins were visualized using enhanced chemiluminesence reagent (Western Lightning Plus-ECL, Perkin Elmer Life Sciences, Waltham, MA). The antibodies bound to membrane were stripped and reprobed with the other antibodies as described165657585960.…”

HCV NS5A Protein Containing Potential Ligands for Both Src Homology 2 and 3 Domains Enhances Autophosphorylation of Src Family Kinase Fyn in B Cells

“…B-lymphoid leukemia BJAB cells were kindly provided from Dr. Satoshi Ishido (RIKEN, Yokohama, Japan) [13] and maintained as described previously [14]. For the stable transfection of BJAB cells, 6 µg of linearized pEF1A-NS5A(Con1)-Myc-His was transfected into 5×10 6 cells/500 µl of cells by electroporation (240 V, 950 µF).…”