Stimulation of immune cell receptors, such as the B cell receptor (BCR), can lead to multiple distinct cellular responses, making these cells excellent models for studying signal plasticity. Singh et al. found that inhibition of either release of calcium from the endoplasmic reticulum (with TMB-8) or uptake of calcium from the extracellular medium (with EGTA, a calcium chelator) produced a global dampening of BCR-dependent phosphorylation, including phosphorylation events that occurred within the first minute of BCR activation. Indeed, one of the events that was inhibited was the phosphorylation of Lyn, a Src family tyrosine kinase, which is believed to be the first enzyme activated in response to BCR ligand binding. This was surprising because calcium signals are downstream events; thus, this result suggested that calcium can feed back on the early signaling events. Formation of reactive oxygen species (ROS) in response to BCR activation occurred within seconds and was inhibited by pharmacological inhibition of protein tyrosine kinase activity or phosphoinositide 3-kinase activity. Importantly, ROS production was also inhibited by TMB-8 or EGTA, and inhibition of ROS production attenuated BCR-stimulated increases in intracellular calcium concentration. These effects were dose-dependent, which is consistent with a graded regulatory mechanism rather than a bistable switch mechanism. ROS can inhibit protein tyrosine phosphatase activity, and pharmacological inhibition of calcium signaling or ROS production suppressed BCR-mediated inhibition of two phosphatases, BCR-associated SHP-1 and CD45, which both exhibit a rapid and transient decrease in activity within seconds of BCR stimulation. Finally, the activity of a murine dual oxidase, DUOX1, may provide a mechanism for the regulation of ROS production by calcium, because enzymes in this family have two EF hand motifs, which are calcium-binding motifs. Using siRNA to suppress DUOX1 expression, the authors showed that ROS and calcium signaling were inhibited and that phosphorylation of Lyn was decreased. The authors suggest that low-intensity activation of the BCR produces an initial calcium signal that feeds back and regulates the amplitude of a rapidly produced ROS signal, which then transiently inhibits protein tyrosine phosphatase activity, thereby controlling the amplitude and kinetics of the BCR signal.
D. K. Singh, D. Kumar, Z. Siddiqui, S. K. Basu, V. Kumar, K. V. S. Rao, The strength of receptor signaling is centrally controlled through a cooperative loop between Ca2+ and an oxidant signal. Cell 121, 281-293 (2005). [Online Journal]