Abstract
The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor Gö6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.
Highlights
Signaling through the B cell antigen receptor (BCR)1 is required throughout B cell development and peripheral matura
B cell receptor (BCR)-induced Phosphorylation of cAMP-response element-binding protein (CREB) Is Dependent on c/novel (Ca2ϩ-independent) PKC (nPKC)—We initially sought to demonstrate the requirement for a c/nPKC isoform(s) in BCR-induced CREB phosphorylation on Ser-133
To assess the efficacy of phorbol 12-myristate 13-acetate (PMA) as it pertains to protein kinase C (PKC) down-regulation, we found that the cellular levels of nPKC␦ and nPKC⑀ were depleted by PMA, but not 4␣-PMA pretreatment (Fig 1A, lanes nPKC␦ and nPKC⑀)
Summary
Signaling through the B cell antigen receptor (BCR)1 is required throughout B cell development and peripheral matura-. In agreement with the data, pretreatment of splenic B cells with PMA alone led to a near complete block in anti-Ig-induced CREB phosphorylation at the time points examined (Fig. 2, lanes pCREB).
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