Analysis of the ubiquitination of Syk at the BCR and stress granules

Renee Lynn Killins, Purdue University


Syk plays a crucial role in B cell growth and maturation and has been implicated in numerous disease states. A cell-permeable, peptide-based probe that is selectively phosphorylated by Syk in comparison to other known tyrosine kinases was developed to study the activation state of Syk in disease models. This peptide probe was tested in in vitro kinase assays as well as in a number of cell lines including DT40 chicken B cells and DG75 Burkitt's lymphoma cells. The accuracy and specificity of the peptide was tested using Syk inhibitors. The peptide could be used successfully to measure changes in Syk activity. Results were confirmed by comparing changes in peptide phosphorylation to those of actual Syk substrates in the cell such as BLNK. The ultimate goal is to develop a clinically useful probe. Therefore, primary splenic B cells from mice were used to test if the peptide substrate could be taken up and used to accurately measure Syk activity in non-immortalized cells. The Syk substrate peptide was able to measure accurately Syk activity in primary cells. The ubiquitination of Syk was investigated to better understand the tight regulation of this tyrosine kinase in cells. Syk ubiquitination persists for 13 minutes following BCR ligation. In cells treated with proteasome inhibitor, Syk ubiquitination persisted longer although it still began to decrease by 20 minutes. These results suggest that Syk ubiquitination results in proteasomal degradation, but also has other outcomes. Following BCR ligation, Syk becomes modified by multiple types of ubiquitin chain linkages and is localized predominantly to the cytoplasm. The overexpression of TRIP results in an increase in K48-linked Syk ubiquitination. An in vitro ubiquitination assay determined that TRIP can directly catalyze Syk ubiquitination. Interestingly, overexpression of ΔRING TRIP, an inactive form of TRIP, also results in an increase in Syk ubiquitination. Thus, TRIP is perhaps involved in the ubiquitination of another E3 of Syk such as TRAF2. TRIP and Syk have previously been found to localize in cytoplasmic puncta resembling stress granules following TNF-&agr; treatment. I have shown for the first time that Syk localizes to stress granules following treatment of cells with sodium arsenite. The activity of Syk is required for its recruitment to these cytoplasmic complexes and the induction of stress granules results in Syk ubiquitination. These studies elucidate TRIP-mediated Syk ubiquitination and implicate a role for Syk ubiquitination in the cellular response to stress.




Geahlen, Purdue University.

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