CD45 MONOCLONAL-ANTIBODIES INHIBIT TCR-MEDIATED CALCIUM SIGNALS, CALMODULIN-KINASE-IV GR-ACTIVATION, AND ONCOPROTEIN-18 PHOSPHORYLATION

The effects of a pan-CD45 mAb (CD45.2) on TCR-mediated signaling pathw ays were investigated in Jurkat T cells. The simultaneous addition of CD45 mAb with an activating OKT3 mAb had little effect on TCR-stimulat ed signals. However, when Jurkat cells were exposed to the CD45 mAb fo r 10 to 20 min before the addition of OKT3, a partial uncoupling of th e TCR from intracellular signals was observed. The maximal increase in intracellular calcium was inhibited 47 +/- 10% (n = 11, range 33-67%) , whereas no inhibition of inositol trisphosphate production was detec ted. The transient TCR-mediated activation of the Ca2(+)/calmodulin-ac tivated kinase IV/Gr was also inhibited by the CD45 mAb, and this was reflected in a 50 to 60% inhibition in the TCR-stimulated generation o f the p21 and p23 phosphoisomers of oncoprotein 18, a Ca2(+)/calmoduli n-activated kinase IV/Gr substrate recently implicated in cell cycle r egulatory events. Oncoprotein 18 is also a substrate for mitogen-activ ated protein kinase, but no inhibition by the CD45 mAb of TCR-triggere d mitogen-activated protein kinase activation was observed. The CD45 m Ab was therefore selective in causing the uncoupling of the TCR from c alcium signals and calcium-regulated events without promoting a genera l inhibition of all TCR-mediated signals. Confocal microscopy revealed that binding of the CD45 mAb caused patching of CD45 molecules at the cell surface and, unexpectedly, a marked redistribution of intracellu lar CD45. However, no change was observed in the total level of CD45 e xpressed at the cell surface. Aggregation of CD45 at the cell surface may result in its sequestration from its tyrosine kinase substrates, w ith a consequent selective uncoupling of the TCR from intracellular si gnaling pathways.