Activation of Lyn kinase and translocation of its PKC6 target regulate phosphorylation in the IL-4-induced alternate pathway for BCR signaling

Abstract B cell signaling for activation via the BCR occurs as an isolated event only in vitro; in real life, BCR signaling takes place within a complex milieu that involves interactions with agents that trigger additional receptors. Chief among these agents is IL-4. We have shown that BCR signaling is reprogrammed by IL-4 receptor engagement, and that this reprogramming involves creation of a new, signalosome-independent, Lyn-dependent alternate signaling pathway out of pre-existing, otherwise noninteracting, elements. A unique aspect of alternate pathway BCR signaling is PKCδ phosphorylation. In dissecting this pathway, we unexpectedly found that IL-4 alone induces phosphorylation and activation of Lyn, and that Lyn immunoprecipitated from IL-4-treated B cells capably phosphorylates PKCδ in a cell-free system, although PKCδ phosphorylation does not occur in the absence of BCR triggering in vivo. This raised the question of why IL-4 alone failed to produce PKCδ phosphorylation. We considered the possibility that Lyn and PKCδ may be spatially separated. As expected, Lyn is located primarily in the membrane fraction, whereas PKCδ is located mainly in the cytosol fraction. However, when anti-Ig follows IL-4 treatment, PKCδ is found in the membrane fraction and phosphorylated. This translocation of PKCδ to the membrane fraction is not affected by loss of Lyn, although PKCδ is not phosphorylated without Lyn. Thus, PKCδ phosphorylation through the alternate pathway appears to consist of several discrete steps: 1.IL-4 induces Lyn phosphorylation and activation; 2. IL-4 produces a change such that PKCδ is translocated from the cytosol to the membrane after BCR engagement; 3. Activated Lyn in the membrane phosphorylates PKCδ in the membrane.