Tyrosine kinase and CAMP-dependent protein kinase activities in CD40-activated human B lymphocytes
In vitro, human B lymphocytes undergo long-term proliferation when activated through CD40, a protein expressed on their cell surface. The nature of CD40dependent signals in proliferating fresh human Epstein-Barr virus-negative B lymphocytes is currently unknown. In this study, a CD40-dependent B cell culture system was used to examine the role of different signal transduction elements. Protein kinase C (PKC) depletion generated by a long-term phorbol 12myristate 13-acetate treatment had weak effects on proliferation. Rather, tyrosine phosphorylation was shown to be directly involved in mediating CD40dependent signals. The use of the protein tyrosine kinase (PTK)-specific inhibitor herbimycin A dramatically decreased cellular proliferation without altering the activity of the human immunodeficiency virus-1 long terminal repeat (HIV-1 LTR), a promoter largely dependent on the binding of nuclear factor xB (NF-xB). In contrast, the CAMP-dependent protein kinase specific inhibitor H-89 totally inhibited HIV-1 LTR activity at a concentration as low as 100 nM without affecting cellular proliferation. Electrophoretic mobility shift assay (EMSA) and supershift assay using an NF-xB binding sequence from the x light chain as a probe, revealed that both p65 (RelA) and c-Re1 were present in CD40stimulated B cells. While PKC depletion did not alter the NF-xB level, treatment of B lymphocytes with H-89 or herbimycin A provoked a decrease in the NF-xB level. These observations establish the importance of different signal transducing pathways leading to CD40 activation of B lymphocytes.