Transphosphorylation of Bruton's Tyrosine Kinase on Tyrosine 551 Is Critical for B Cell Antigen Receptor Function

Abstract

is essential for BCR function and that this phosphorylation is mediated through the concerted actions of Lyn and Syk.The B cell antigen receptor (BCR) 1 is composed of surface immunoglobulin noncovalently associated with a pair of Ig␣/ Ig␤ disulfide-linked heterodimers, which are essential for signal transduction. Stimulation of the BCR induces the enzymatic activation and tyrosine phosphorylation of three distinct families of nonreceptor cytoplasmic protein tyrosine kinases (PTKs), the Src family, Syk, and Btk. The Src family kinases are rapidly activated after BCR engagement, and their activation correlates with the initial tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif on the BCR Ig␣ and Ig␤ subunits (reviewed in Refs. 1-4).Temporally, activation of Src family kinases is followed by Btk and Syk (5). This sequential activation potentially places Btk and Syk downstream of Src family kinases. Utilizing cooverexpression system in fibroblasts and COS cells, it has been demonstrated that Lyn transphosphorylates Btk on Tyr 551 in the catalytic domain, a site homologous to the Src family kinase consensus autophosphorylation site (6, 7). This results in a 5-10-fold increase in Btk enzymatic activity (7). The increase in activity also leads to increased autophosphorylation at Tyr 223 in the SH3 domain of Btk (8). The identical phosphopeptides were generated after cross-linking of the BCR, indicating that these sites are also tyrosine phosphorylated in B cells (7 EXPERIMENTAL PROCEDURESCells, Antisera, and DNA Transfection-DT40 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine. Anti-chicken IgM mAb M4 and anti-phospholipase C (PLC)-␥2 Ab were described previously (10). The anti-phosphotyrosine mAb (4G10) and anti-T7 mAb were obtained from Upstate Biotechnology, Inc. and Novagen, respectively. T7-tagged and mutant Btk cDNAs were created by polymerase chain reaction, and the resulting constructs were confirmed by DNA sequencing. These cDNAs were cloned into pApuro expression vector (10). For DNA transfection into DT40 cells, DNA was linearized, electroporated, and selected in the presence of puromycin (0.5 g/ml). The expression of Btk was analyzed by Western blotting.Immunoprecipitation and Immunoblot Analysis-DT40 cells were stimulated by mAb M4 for indicated time. Cells were solubilized in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris, pH 7.5, 1 mM EDTA) containing 50 mM NaF, 10 M molybdate, and 0.2 mM sodium vanadate supplemented with protease inhibitors described previously (10). Cell lysates were sequentially incubated (1 h at 4°C for each incubation with Ab and protein A-Sepharose). For immunoblotting, samples were separated on SDS-PAGE and transferred to nitrocellulose membrane (Amersham Corp.). Filters were incubated with mAb 4G10 or anti-T7 mAb. After washing, filters were developed using a sheep anti-mouse IgG Ab conjugated to horseradish peroxidase and enhanced chemiluminescence (ECL).For in v...

Function of Bruton’s Tyrosine Kinase during B Cell Development Is Partially Independent of Its Catalytic Activity

“…Taken together, these results indicate that Y223 autophosphorylation-dependent interactions are not essential for B cell development and mature B cell function. This is consistent with previous observations that reconstitution of Btk-deficient chicken DT 40 B cells with Y223F and WT Btk resulted in a similar rescue of BCR-induced PLC-␥ phosphorylation and calcium flux (39). Our findings may also explain the remarkable absence of missense mutations in the Btk SH3 domain in a total of 155 missense mutations identified to date in XLA families (38), while on the basis of SH3 domain size ϳ10 of these should be located within the SH3 domain.…”

Activation Mechanism and Steady State Kinetics of Bruton's Tyrosine Kinase

“…Tyr 551 is a well known phosphorylation site of BTK. However, most reports have attributed phosphorylation of this site within B cells to the Lyn kinase rather than autophosphorylation (14,15,26). Hence, the robust autophosphorylation observed here is somewhat surprising.…”

BLNK mediates Syk-dependent Btk activation

“…Coexpression experiments in B cells and fibroblasts have demonstrated that Lyn transphosphorylates the tyrosine 551 residue (Y551) in the Btk catalytic domain, which is the critical residue for the enhancement of Btk catalytic activity (5,6). Y551 in Btk is also phosphorylated after BCR crosslinking, which indicates that the phosphorylation of Y551 is also important for BCR signaling (6,7). In addition to the Src-family PTKs, recent findings have indicated that Syk is also involved in Btk activation.…”

“…In addition to the Src-family PTKs, recent findings have indicated that Syk is also involved in Btk activation. A genetic dissection experiment on the DT40 B cell line demonstrated that the BCR-induced tyrosinephosphorylation of Btk is significantly reduced in Sykdeficient cells as well as in Lyn-deficient cells (7). Furthermore, Btk phosphorylation is almost completely eliminated in Lyn͞Syk double-deficient cells (7).…”