Modulation of human neutrophil responses to CD32 cross-linking by serine/threonine phosphatase inhibitors: Cross-talk between serine/threonine and tyrosine phosphorylation

The interplay between serine/threonine and tyrosine phosphorylation mas stu died in human neutrophils, The direct effects of calyculin and okadaic acid , potent inhibitors of PP1 and PP2A serine/threonine phosphatases, on the p atterns of neutrophil phosphorylation, and their effects on the responses o f neutrophils to CD32 cross-linking were monitored. After a 2-min incubatio n with 10(-6) M calyculin, a transient tyrosine phosphorylation of a subset of proteins, among which Cbl and Syk, was observed. After a longer incubat ion (>5 min) with calyculin, concomitant with an accumulation of serine and threonine phosphorylation, neutrophil responses to CD32 cross-linking were selectively altered, Tyrosine phosphorylation of Cbl in response to CD32 c rosslinking was inhibited by calyculin, and this inhibition was linked with a slower electrophoretic mobility of Cbl as a consequence of its phosphory lation on serine/threonine residues. However, tyrosine phosphorylation of S yk and of the receptor itself were not affected. Furthermore, the mobilizat ion of intracellular calcium stimulated by CD32 cross-linking was totally a brogated by calyculin. Finally, the stimulation of superoxide production ob served in response to CD32 cross-linking was enhanced in calyculin-treated cells. These results suggest that serine/threonine phosphorylation events r egulate the signaling pathways activated by CD32 cross-linking in neutrophi ls and identify a novel mechanism of modulation of the functional responsiv eness of human neutrophils to CD32 cross-linking.