Abstract
T cell antigen receptor (TCR) and B cell antigen receptor (BCR) signaling are initiated and tightly regulated by Src-family kinases (SFKs). SFKs positively regulate TCR signaling in naïve T cells but have both positive and negative regulatory roles in BCR signaling in naïve B cells. The proper regulation of their activities depends on the opposing actions of receptor tyrosine phosphatases CD45 and CD148 and the cytoplasmic tyrosine kinase C-terminal Src kinase Csk. Csk is a major negative regulator of SFKs. Using a PP1-analog-sensitive Csk (CskAS) system, we have previously shown that inhibition of CskAS increases SFK activity, leading to augmentation of responses to weak TCR stimuli in T cells. However, the effects of Csk inhibition in B cells were not known. In this study, we surprisingly found that inhibition of CskAS led to marked inhibition of BCR-stimulated cytoplasmic free calcium increase and Erk activation despite increased SFK activation in B cells, contrasting the effects observed in T cells. Further investigation revealed that acute CskAS inhibition suppressed BCR-mediated phosphatidylinositol 3,4,5-trisphosphate (PIP3) production in B cells. Restoring PIP3 levels in B cells by CD19 cross-linking or SHIP1 deficiency eliminated the negative regulatory effect of CskAS inhibition. This reveals the critical role of Csk in maintaining an appropriate level of SFK activity and regulating PIP3 amounts as a means of compensating for SFK fluctuations to prevent inappropriate B cell activation. This regulatory mechanism controlling PIP3 amounts may also contribute to B cell anergy and self-tolerance.
Highlights
T cell antigen receptor (TCR) and B cell antigen receptor (BCR) signaling are initiated and tightly regulated by Src-family kinases (SFKs)
We observed marked inhibition of BCR-downstream signaling due to associated impairment of the phosphatidylinositol-trisphosphate pathway. These results reveal the critical importance of maintaining a proper amount of SFK activity in quiescent B cells for appropriate BCR-dependent responses, which may be critical for naïve B cell unresponsiveness to self-antigens to maintain peripheral tolerance
As early as 30 s after adding 3-iodobenzyl analog of the kinase inhibitor PP1 (3IB-PP1) to CskAS B cells we observed a rapid decrease in phosphorylation of the inhibitory-tail tyrosines on SFKs and strong increases in SFKs activation-loop tyrosine phosphorylation (Fig. 1 A and B)
Summary
T cell antigen receptor (TCR) and B cell antigen receptor (BCR) signaling are initiated and tightly regulated by Src-family kinases (SFKs). Restoring PIP3 levels in B cells by CD19 cross-linking or SHIP1 deficiency eliminated the negative regulatory effect of CskAS inhibition This reveals the critical role of Csk in maintaining an appropriate level of SFK activity and regulating PIP3 amounts as a means of compensating for SFK fluctuations to prevent inappropriate B cell activation. Lyndeficient B cells are hyperproliferative and exhibit increased Erk phosphorylation and increased calcium in response to BCR stimulation, which reveals that Lyn plays a nonredundant role in activating downstream ITIMs and functions as a net negative regulator of BCR signaling [7]. When phosphorylated by Src-family kinases (SFKs), ITIM receptors recruit SH2 domain-containing protein (SHPs) and lipid phosphatases (SHIPs) that directly dephosphorylate various signaling molecules [1].
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