Proteasome-dependent regulation of Syk tyrosine kinase levels in human basophils

[摘要] Background: In human basophils, FcepsilonRI signal initiation, leading to histamine release, relies on activation of Syk protein tyrosine kinase. Basophils from approximately 10%, of unselected donors do not degranulate in response to FcepsilonRI crosslinking. Their unresponsiveness has been linked to the absence of Syk protein despite apparently normal levels of Syk mRNA. Objective: The aim of this study was to explore pathways of Syk protein degradation as a possible posttranslational mechanism for downregulating Syk protein levels in human basophils and other leukocytes. Methods: Highly purified basophils, lymphocytes, and monocytes were incubated in the presence or absence of a panel of cell-permeable inhibitors of proteolytic degradation pathway(s). Subsequently, the protein level of Syk tyrosine kinase was determined by means of Western blotting. In vitro assays were conducted through use of immunoprecipitated basophil Syk and a rabbit reticulocyte lysate system. Results: Three inhibitors of proteasome-mediated degradation-PSI, lactacystin, and ALLN-substantially increased Syk levels in releaser basophils and restored Syk expression in nonreleaser basophils. Caspase inhibitors were less effective, and inhibitors of calpain-mediated proteolysis had no effect. Among other leukocytes tested, only naive CD4(+) T cells had more Syk after proteasome inhibitor treatment. In vitro ubiquitination assays demonstrated that Syk is readily ubiquitinated in vitro and also that Syk ubiquitination is associated with a substantial decrease in total levels of Syk protein. Conclusion: These data provide evidence for a ubiquitin/proteasome-dependent mechanism that contributes to Syk regulation in human basophils and might also be relevant to naive T cells. Understanding this regulatory pathway might lead to strategies for suppressing allergic inflammation while preserving essential Syk-mediated functions in other hematopoietic cells.

[发布日期] 2002-09-01

[关键词] basophil;Lyn;Syk;Fc epsilon RI;proteasome;ubiquitin