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 elements in several cascades coupling immune receptors to intracellular responses. The critical role of these kinases in promoting the propagation of intracellular signaling requires a tight regulation of their activity, thus the existence of a negative feedback loop regulating their expression can be hypothesized. Herein, we have investigated whether ubiquitin-dependent proteolysis could be a mechanism responsible for controlling the fate of Syk and 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 confirmed the identity of the major in vivo ubiquitinated kinase species by performing an in vitro ubiquitination assay. In addition, we found that after CD16 stimulation, ubiquitinated forms of Syk and ZAP-70 associate with the receptor 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 ubiquitinated 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 mechanism for attenuating the propagation of intracellular signaling initiated by immune receptor engagement.
Paolini
R
R
Department of Experimental Medicine and Pathology, Institute Pasteur-Fondazione Cenci Bolognetti, University La Sapienza, Rome, Italy. rossella.paolini@uniroma1.it
Molfetta
R
R
Piccoli
M
M
Frati
L
L
Santoni
A
A
D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D005246:Feedback; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology; D046911:Macromolecular Substances; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017452:Receptors, IgG / Q000276:immunology*; D015398:Signal Transduction / Q000502:physiology*; D000072377:Syk Kinase; D014452:Ubiquitins / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase
D004792:Enzyme Precursors; D007074:Immunoglobulin G; D047908:Intracellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011480:Protease Inhibitors; D021122:Protein Subunits; D017452:Receptors, IgG; D014452:Ubiquitins; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C078846:epoxomicin