Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation

Abstract

Bruton's tyrosine kinase (Btk) is essential for B‐lineage development and represents an emerging family of non‐receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B‐cell antigen receptor (BCR) cross‐linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk‐deficient human B‐cell lines. Btk and phospholipase Cγ (PLCγ) co‐expression resulted in tyrosine phosphorylation of PLCγ and required the same Btk domains as those for Btk‐dependent calcium influx. Receptor‐dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)‐sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg‐sensitive, IP3‐gated calcium store(s) that regulates store‐operated calcium entry. Overexpression of dominant‐negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co‐cross‐linking of the BCR and the inhibitory receptor, FcγRIIb1, completely abrogated Btk‐dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.

The tyrosine kinase Syk is required for light chain isotype exclusion but dispensable for the negative selection of B cells

“…[11]. A similar profile of calcium flux to that described here was observed in A20 cells expressing a dominant negative form of Syk [33].…”

Reduced Formation of Phosphatidic Acid upon B‐Cell Receptor Triggering of Mouse B‐Lymphocytes Lacking Bruton's Tyrosine Kinase

“…3B). Similar ®ndings were reported in the XLA cell lines [22], while in the Btk de®cient DT 40 cells, the BCR-induced tyrosine phosphorylation of PLCg2 was reduced [20]. The fact that several studies have suggested that PLCg is a substrate for Btk [20,22,23], could argue for compensating kinase elements (i.e.…”

“…However, our failure to 34 J. Forssell et al detect PEt formation upon BCR stimulation, although readily detectable using phorbol esters, argues that the PA we detected in our experimental set-up was not derived from a PLD-activity, but rather from PLC mediated PI-hydrolysis. Reduced PI-turnover is also detected in human XLA cell-lines [22], xid B cells [24] and TCR stimulated mouse CD4 T cells de®cient for Itk (another member of the Btk family) [50], suggesting that regulation of PI-hydrolysis could be a general property of the Btk/Tec/Itk family kinases.…”

“…Tec) operating in the Btk M B cells, especially considering the modest reduction in calcium in¯ux. Alternatively, PLCg bulk tyrosine phosphorylation may mask reduction at a critical site, as suggested by Fluckiger et al [22]. In the study using xid B cells the issue of PLCg tyrosine phosphorylation was never addressed, but a qualitative or quantitative defect in a phospholipase was suggested as a possible explanation to the reduced calcium¯uxes detected [24].…”

“…An intimate link between PI3-kinase activity and calcium¯uxes has recently been documented [34, 38±41] and studies in human and DT40 cell lines have indicated that interactions between Btk and PI(3,4,5)P 3 are critical for extra-cellular calcium in¯ux, identifying Btk as a target of Ship inhibitory signals [22,23,42]. Additionally, membrane-targeted Btk could rescue calcium signalling when PI3-kinase was inhibited in anti-Ig-stimulated DT40 cells either with wortmannin or by cocross-linking the FcgRIIb-receptor to the BCR [42].…”