Ubiquitination and degradation of Syk and ZAP-70 protein tyrosine kinases in human NK cells upon CD16 engagement

Syk and ZAP-70 nonreceptor protein tyrosine kinases (PTKs) are essential el ements in several cascades coupling immune receptors to intracellular respo nses. The critical role of these kinases in promoting the propagation of in tracellular signaling requires a tight regulation of their activity, thus t he existence of a negative feedback loop regulating their expression can be hypothesized. Herein, we have investigated whether ubiquitin-dependent pro teolysis could be a mechanism responsible for controlling the fate of Syk a nd ZAP-70 after their immunoreceptor-induced activation. We found that both Syk and ZAP-70 become ubiquitinated in response to aggregation of the low affinity Fc receptor for IgG (CD16) on human natural killer cells. We confi rmed the identity of the major in vivo ubiquitinated kinase species by perf orming an in vitro ubiquitination assay. In addition, we found that after C D16 stimulation, ubiquitinated forms of Syk and ZAP-70 associate with the r eceptor complex. After CD16 engagement, we also observed a decrease in the stability of Syk and ZAP-70 PTKs that is counteracted by pretreatment with either proteasome or lysosomal inhibitors. Moreover, in the presence of the proteasome inhibitor, epoxomicin, we observed an accumulation of ubiquitin ated forms of both kinases. Our findings provide evidence of ligand-induced ubiquitination of nonreceptor PTKs belonging to the Syk family and propose the ubiquitin-dependent proteasome-mediated degradation pathway as a mecha nism for attenuating the propagation of intracellular signaling initiated b y immune receptor engagement.