Perturbed regulation of ZAP-70 and sustained tyrosine phosphorylation of LAT and SLP-76 in c-Cbl-deficient thymocytes.
Recent studies indicate that c-Cbl and its oncogenic variants can modulate the activity of protein tyrosine kinases. This finding is supported by studies showing that c-Cbl interacts directly with a negative regulatory tyrosine in ZAP-70, and that the levels of tyrosine-phosphorylated ZAP-70 and numerous other proteins are increased in TCR-stimulated thymocytes from c-Cbl-deficient mice. Here, we demonstrate that this enhanced phosphorylation of ZAP-70 and that of two substrates, LAT and SLP-76, is not due to altered protein levels but is the consequence of two separate events. First, we find increased expression of tyrosine-phosphorylated TCRzeta chain in c-Cbl-deficient thymocytes, which results in a higher level of zeta-chain-associated ZAP-70 that is initially accessible for activation. Thus, more ZAP-70 is activated and more of its substrates (LAT and SLP-76) become tyrosine-phosphorylated after TCR stimulation. However, an additional mechanism of ZAP-70 regulation is evident at a later time poststimulation. At this time, ZAP-70 from both normal and c-Cbl-/- thymocytes becomes hyperphosphorylated; however, only in normal thymocytes does this correlate with ZAP-70 down-regulation and a diminished ability to phosphorylate LAT and SLP-76. In contrast, c-Cbl-deficient thymocytes display altered phosphorylation kinetics, for which LAT phosphorylation is increased and SLP-76 phosphorylation is sustained. Thus, the ability to down-regulate the phosphorylation of two ZAP-70 substrates is impaired in c-Cbl-/- thymocytes. These findings provide evidence that c-Cbl is involved in the negative regulation of the phosphorylation of LAT and SLP-76 by ZAP-70.
Thien
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CB
Department of Pathology, University of Western Australia, Queen Elizabeth II Medical Center, Nedlands.
Bowtell
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DD
Langdon
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WY
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013601:T-Lymphocytes / Q000201:enzymology* / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains / Q000276:immunology*
D048868:Adaptor Proteins, Signal Transducing; D015704:CD4 Antigens; D002352:Carrier Proteins; C491734:Lat protein, mouse; D008565:Membrane Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D011948:Receptors, Antigen, T-Cell; C092947:SLP-76 signal Transducing adaptor proteins; C054979:antigen T cell receptor, zeta chain; D014443:Tyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse; C493849:Cbl protein, mouse