TRANSCRIPTIONAL REGULATION OF THE JUNB GENE IN B-LYMPHOCYTES - ROLE OF PROTEIN-KINASE-A AND A MEMBRANE IG-REGULATED PROTEIN PHOSPHATASE

We have examined herein whether membrane Ig (mIg) stimulates junB tran scription through a protein kinase A (PKA)-dependent or PKA-independen t pathway, PKA phosphotransferase activity was not increased following mIg cross-linking of Bal17 B cells, However, junB transcriptional act ivation was dependent upon PKA activity, as evidenced by inhibition of goat anti-mouse IgM-stimulated junB promoter-chloramphenicol acetyltr ansferase reporter gene activity in transfected Bal17 B cells treated with the PKA inhibitor H-89, mIg-stimulated junB promoter-chlorampheni col acetyltransferase activity was also blocked in B cells expressing a specific PKA inhibitor peptide, whereas in vivo expression of an ina ctive PKA inhibitor peptide variant was not inhibitory, Expression of a mutant cAMP response element binding protein (CREB) containing an in activated kinase A phosphoacceptor site at Ser(133) reduced mIg-stimul ated junB transcription, Okadaic acid increased CREB1 phosphorylation at Ser(133) and junB transcriptional activation, suggesting the action of protein phosphatase-1 (PP-1) or -2A (PP-2A). Extracts from unstimu lated B cells exhibited phosphatase activity against an in vitro PKA-p hosphorylated peptide containing the Ser(133) phosphoacceptor site, Th e involvement of a phosphatase activity in regulating mIg-stimulated j unB transcription is supported by our finding that extracts from goat anti-mouse IgM-stimulated B cells exhibited a significantly reduced le vel of Ser(133) phosphatase activity, Hence, the level of CREB1 phosph orylation is governed by the balance between PKA and phosphatase activ ities, junB transcriptional activation results in part from mIg signal s that negatively regulate a CREB1-targeted PP-1 or PP-2A activity.