B-Cell Receptor- and Phorbol Ester-Induced NF-κB and c-Jun N-Terminal Kinase Activation in B Cells Requires Novel Protein Kinase C's

Abstract

Antigen receptor signaling is known to activate NF-κB in lymphocytes. While T-cell-receptor-induced NF-κB activation critically depends on novel protein kinase C θ (PKCθ), the role of novel PKCs in B-cell stimulation has not been elucidated. In primary murine splenic B cells, we found high expression of the novel PKCs δ and ɛ but only weak expression of the θ isoform. Rottlerin blocks phorbol ester (phorbol myristate acetate [PMA])- or B-cell receptor (BCR)-mediated NF-κB and c-Jun N-terminal kinase (JNK) activation in primary B and T cells to a similar extent, suggesting that novel PKCs are positive regulators of signaling in hematopoietic cells. Mouse 70Z/3 pre-B cells have been widely used as a model for NF-κB activation in B cells. Similar to the situation in splenic B cells, rottlerin inhibits BCR and PMA stimulation of NF-κB in 70Z/3 cells. A derivative of 70Z/3 cells, 1.3E2 cells, are defective in NF-κB activation due to the lack of the IκB kinase (IKKγ) protein. Ectopic expression of IKKγ can rescue NF-κB activation in response to lipopolysaccharides (LPS) and interleukin-1β (IL-1β), but not to PMA. In addition, PMA-induced activation of the mitogen-activated protein kinase JNK is blocked in 1.3E2 cells, suggesting that an upstream component common to both pathways is either missing or mutated. Analysis of various PKC isoforms revealed that exclusively PKCθ was absent in 1.3E2 cells while it was expressed in 70Z/3 cells. Stable expression of either novel PKCθ or -δ but not classical PKCβII in 1.3E2 IKKγ-expressing cells rescues PMA activation of NF-κB and JNK signaling, demonstrating a critical role of novel PKCs for B-cell activation.

ACKNOWLEDGMENTS

We thank Gottfried Baier for providing us with PKCθ and PKCδ, Harald Mischak for the gift of PKCβΙΙ, and Robert Newton for the 6xNF-κBluc constructs. We thank Carol Sibley for the gift of the 70Z/3 and 1.3E2 cells. Furthermore, we thank Erika Scharschmidt for excellent technical assistance, Benjamin Mordmüller for purification of GstJun1–79, and Susanne Preiss for the help with the lymphocyte purification.