Regulation of glycogen synthase kinase-3 (GSK-3) and β-catenin by CD40 in B lymphocytes

Antibodies are specialized molecules produced by B-lymphocytes to combat infection by binding to and targeting infectious agents for destruction. To produce antibodies, B-cells require signals from multiple receptors, including the B-cell antigen receptor (BCR) and CD40. β-catenin is a transcriptional co-activator that regulates important proproliferative genes. Phosphorylation of β-catenin by glycogen synthase kinase-3 (GSK- 3) in resting B cells targets β-catenin for proteasome-mediated degredation, keeping levels of β-catenin low. B C R engagement results in β-catenin upregulation by phosphorylating GSK-3 on negative regulatory sites and inhibiting GSK-3 activity. β-catenin then localizes to the nucleus, and activates transcription. I show that CD40 also regulates GSK-3, but does so through a different intracellular signaling pathway than the BCR. I show that CD40 induces the phosphorylation of GSK-3 on negative regulatory sites via a pathway that is independent of phosphatidylinositol-3 kinase (PI3K) and Akt, but requires the activity of MEK-1. Also, I used a reporter gene assay to show that BCR and CD40 stimulation act together to induce a greater level of β-catenin-dependent transcription than BCR stimulation alone. Two alternate hypotheses are possible to explain this effect. Either CD40 enhances the upregulation and nuclear localization of β-catenin caused by the BCR, or CD40 signaling upregulates β-catenin binding partners. The binding partners of (3-catenin are transcription factors of the TCF/LEF family. Recent reports showed that in response to CD40 signaling LEF-1 mRNA increases. This suggests that the synergistic increase in β-catenin mediated transcription could be due to CD40 induced upregulation of β-catenin binding partners. I used quantitative real-time polymerase chain reaction to show that one possible mechanism for the CD40-mediated increase of β-catenin-mediated transcription is CD40 induced upregulation of TCF-1 mRNA. The GSK-3/β-catenin/TCF-1 pathway may be a key point of integration between B C R and CD40 signaling which regulates B cell differentiation. In addition, GSK-3 and β-catenin have been implicated in the development of B-cell malignancies. Greater understanding of the interactions of these key regulatory pathways in the activation of B-cells is essential to our understanding of how humoral immune responses clears infection, and could provide insight into the development of B-cell leukemias.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat