h-89 and Lymphoma--B-Cell

Signaling through the B cell antigen receptor (BCR) is a key determinant in the regulation of B cell physiology. Depending on additional factors, such as microenvironment and developmental stage, ligation of the BCR can trigger B lymphocyte activation, proliferation, or apoptosis. The regulatory mechanisms determining B cell apoptosis and survival are not completely known. Using the murine B lymphoma cell line WEHI-231 as a model system, we investigated the role of Bad phosphorylation, a pro-apoptotic member of the Bcl-2 family, in anti-IgM mediated apoptosis. For apoptotic analysis we focused in particular on the mitochondrial potential (deltapsi(m)) collapse which has been reported as a rate-limiting step in the BCR-induced cell death of immature B lymphocytes. Bad phosphorylation at serine 112, 136 and 155 was found in WEHI-231 cell control cultures and its hypophosphorylation on the three sites correlated with the appearance of apoptosis when cross-linking surface IgM. Furthermore, treatment of cells with specific PK inhibitors known to be involved in serine phosphorylation of Bad (LY294002 for PI3K and H-89 for PKA) mimiced or enhanced BCR-induced cell death. These results strongly suggest that regulation of Bad phosphorylation plays an active role in mediating anti-IgM-induced apoptosis of immature B cells.

Topics: 14-3-3 Proteins; Animals; Apoptosis; B-Lymphocytes; bcl-Associated Death Protein; bcl-X Protein; Carrier Proteins; Cells, Cultured; Chromones; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Immunoglobulin M; Isoquinolines; Lymphoma, B-Cell; Mice; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, B-Cell; Serine; Sulfonamides; Tyrosine 3-Monooxygenase

We have examined herein whether membrane Ig (mIg) stimulates junB transcription through a protein kinase A (PKA)-dependent or PKA-independent pathway. PKA phosphotransferase activity was not increased following mIg cross-linking of Bal17 B cells. However, junB transcriptional activation was dependent upon PKA activity, as evidenced by inhibition of goat anti-mouse IgM-stimulated junB promoter-chloramphenicol acetyltransferase reporter gene activity in transfected Bal17 B cells treated with the PKA inhibitor H-89. mIg-stimulated junB promoter-chloramphenicol acetyltransferase activity was also blocked in B cells expressing a specific PKA inhibitor peptide, whereas in vivo expression of an inactive PKA inhibitor peptide variant was not inhibitory. Expression of a mutant cAMP response element binding protein (CREB) containing an inactivated kinase A phosphoacceptor site at Ser133 reduced mIg-stimulated junB transcription. Okadaic acid increased CREB1 phosphorylation at Ser133 and junB transcriptional activation, suggesting the action of protein phosphatase-1 (PP-1) or -2A (PP-2A). Extracts from unstimulated B cells exhibited phosphatase activity against an in vitro PKA-phosphorylated peptide containing the Ser133 phosphoacceptor site. The involvement of a phosphatase activity in regulating mIg-stimulated junB transcription is supported by our finding that extracts from goat anti-mouse IgM-stimulated B cells exhibited a significantly reduced level of Ser133 phosphatase activity. Hence, the level of CREB1 phosphorylation is governed by the balance between PKA and phosphatase activities. junB transcriptional activation results in part from mIg signals that negatively regulate a CREB1-targeted PP-1 or PP-2A activity.

Topics: Animals; Antibodies, Anti-Idiotypic; B-Lymphocytes; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Genes, jun; Isoquinolines; Lymphoma, B-Cell; Mice; Peptides; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 1; Proto-Oncogene Proteins c-jun; Receptors, Antigen, B-Cell; Serine; Sulfonamides; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured

We previously reported that cross-linking surface immunoglobulin (sIg) leads to induction of the transcription factor CREB in B lymphocytes through phosphorylation at Ser133, despite the lack of an increase in cAMP. Further, cAMP-raising agents fail to induce CREB Ser133 phosphorylation and CRE-dependent gene expression in these cells, which differs sharply from the situation in PC12 rat pheochromocytoma cells where CREB responds to elevation of cAMP through the activity of protein kinase A. In this study, we characterized the signal transduction pathways leading from sIg engagement to CREB activation. By using specific inhibitors for protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), and protein kinase A (PKA), we found that anti-Ig-induced CREB Ser133 phosphorylation depends on PKC, but does not require activation of PKA or CaM kinase II. The differential responsiveness of CREB to forskolin in PC12 cells and BAL-17 B cells may relate to the more marked elevation of cAMP in the former as opposed to the latter; however, high concentrations of dbcAMP which should readily enter B cells and artificially increase cAMP levels still failed to induce CREB Ser133 phosphorylation, even in conjunction with a phosphodiesterase inhibitor. Taken together, the cAMP/PKA pathway does not appear to be as active a contributor to CREB phosphorylation in B lymphocytes as in PC12 cells, and does not appear to be involved in sIg-induced, PKC-dependent, CREB activation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Antibodies, Anti-Idiotypic; Antigens, CD; B-Lymphocytes; B7-2 Antigen; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation; Immunoglobulins; Ionomycin; Isoquinolines; Lymphoma, B-Cell; Membrane Glycoproteins; Mice; Molecular Sequence Data; Phosphorylation; Piperazines; Protein Kinase C; Rats; Receptors, Antigen, B-Cell; Signal Transduction; Staurosporine; Sulfonamides