SYK Is Upstream of Phosphoinositide 3-Kinase in B Cell Receptor Signaling

Laurie O Beitz,David A Fruman,Tomohiro Kurosaki,Lewis C Cantley,Andrew M Scharenberg

doi:10.1074/jbc.274.46.32662

Abstract

We have recently demonstrated that the D3-phosphoinositide phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) is critical for producing sustained calcium signals through its role in promoting the function of TEC family tyrosine kinases such as Bruton's tyrosine kinase. Although PtdIns-3,4,5-P(3) can potentially be synthesized by any of several types of phosphoinositide 3-kinases (PI3Ks), B cell receptor (BCR)-induced PtdIns-3,4,5-P(3) production is thought to occur primarily through the activation of the class Ia (p85/p110) PI3Ks. This process has been proposed to be mediated by an interaction between the Src family kinase LYN and the p85 subunit of PI3K and/or through p85 membrane recruitment mediated by CBL and/or CD19. However, calcium signaling and other PI3K-dependent signals are relatively preserved in a LYN kinase-deficient B lymphocyte cell line, suggesting that an alternative pathway for PI3K activation exists. As SYK/ZAP70 kinases are upstream from many BCR-initiated signaling events, we directly analyzed SYK-dependent accumulation of both PtdIns-3,4,5-P(3) and PtdIns-3,4-P(2) in B cell receptor signaling using both dominant negative and genetic knockout approaches. Both methods indicate that SYK is upstream of, and necessary for, a significant portion of BCR-induced PtdIns-3,4, 5-P(3) production. Whereas CD19 does not appear to be involved in this SYK-dependent pathway, the SYK substrate CBL is likely involved as the dominant negative SYK markedly attenuates CBL tyrosine phosphorylation and completely blocks the BCR-dependent association of CBL with p85 PI3K.

Highlights

Engagement of surface immunoglobulin (BCR)1 on B cells results in early signaling events including tyrosine phosphorylation of ITAM motifs, activation of the non-receptor protein tyrosine kinases LYN and SYK, and calcium mobilization
These findings suggest that whereas B cell receptor (BCR)-inducible phosphoinositide 3-kinases (PI3Ks) activation may occur through p85 PI3K association with SRC family kinases, it can proceed through SRC kinase-independent routes
In the A20 B cell system, we expressed a dominant negative form of SYK (SYKT) that blocks the interaction of endogenous SYK with BCR ITAMs, resulting in a substantial, but not complete, inhibition of SYK functions such as calcium mobilization (Fig. 1A, this represents a typical degree of inhibition for the samples used in the D3phosphoinositide analyses)

Summary

Introduction

Engagement of surface immunoglobulin (BCR)1 on B cells results in early signaling events including tyrosine phosphorylation of ITAM motifs, activation of the non-receptor protein tyrosine kinases LYN and SYK, and calcium mobilization. PtdIns-3,4,5-P3 can potentially be synthesized by any of several types of phosphoinositide 3-kinases (PI3Ks), B cell receptor (BCR)-induced PtdIns3,4,5-P3 production is thought to occur primarily through the activation of the class Ia (p85/p110) PI3Ks. This process has been proposed to be mediated by an interaction between the Src family kinase LYN and the p85 subunit of PI3K and/or through p85 membrane recruitment mediated by CBL and/or CD19.

Results

Conclusion