PROTEIN-TYROSINE KINASE ACTIVATION AND PROTEIN-KINASE-C TRANSLOCATION ARE FUNCTIONAL COMPONENTS OF CD40 SIGNAL-TRANSDUCTION IN RESTING HUMAN B-CELLS

CD40 is a 47kD glycoprotein expressed on all B cells that plays an important role in B cell development and activation. Previous investigations have focused on signal transduction events in activated B cells and B cell lines, and little information is available regarding CD40 signal transduction in resting, normal B cells. Because CD40 plays a critical role in the regulation and activation of resting B cells, we studied the signaling mechanisms involved in functional responses to CD40 ligation in these cells. Treatment of dense tonsil B cells with either protein tyrosine kinase (PTK) or protein kinase C (PKC) inhibitors, but not with an inhibitor of cyclic nucleotide dependent kinases, resulted in abrogation of CD40)-mediated cell adhesion, suggesting a role for both PTK and PKC in CD40-mediated B cell adhesion. Direct evidence for CD40-mediated PTK activation was demonstrated by increased tyrosine phosphorylation as detected by anti-phosphotyrosine Western blots of cell lysates prepared from dense resting tonsil B cells stimulated with biotinylated anti-CD40 monoclonal antibody plus avidin. CD40 engagement also resulted in PKC activation, as detected by translocation of cytosolic PKC activity to the membrane-bound compartment. CD40-mediated PKC translocation was dependent on PTK activation, whereas PTK activity induced by CD40 ligation was independent of PKC activity, suggesting that PTK activation precedes PKC activation in resting B cells stimulated with anti-CD40 mAb. The results of our experiments identify PTK and PKC activation as components of CD40 signal transduction in normal, resting B cells and establish a functional role for these events.