Detection of Ubiquitination on Syk and Documenting Syk Stability

Post-translational modifications regulate the activities of proteins important to numerous diseases. Spleen Tyrosine Kinase (Syk) is particularly interesting to researchers because it modifies many targets and plays multiple roles in regulating cells in our bodies and its abnormal modifications may contribute to cancer, Alzheimer’s disease and allergies. In an attempt to study these modifications of Syk, we first looked at detecting ubiquitination on Syk protein. Ubiquitin, a small 8 kDa molecule, attaches to lysine residues on protein. The attachment of ubiquitin to Syk may cause Syk to either propagate signals onwards to activate other proteins or signal it to undergo proteasomal degradation. To detect ubiquitination of Syk, B cell lymphoma DG75 with endogenous Syk expression was electroporated with HA-tagged Ubiquitin expression vector to introduce the ubiquitin molecule into the cells. Immunoprecipitation of Syk was performed to isolate the total Syk and to visualize the ubiquitination by Western Blot with anti-HA antibody. When cells were treated with Cyclohexamide (CHX), a protein translation inhibitor, we did not observe significant decrease of Syk in protein level, indicating that Syk is an exceptionally stable protein with a half-life longer than 72 hours. Upon treatment of cells with both CHX and MG132, a proteasome inhibitor, we reproducibly observed a detectable accumulation of Syk protein in 24 hours. The established technique will not only facilitate the study of the impact of ubiquitination on Syk in signal transduction, it also will lead us to identify the potential significance of ubiquinated lysine residues on Syk in cellular function

Spleen tyrosine kinase-mediated autophagy is required for epithelial-mesenchymal plasticity and metastasis in breast cancer

The ability of breast cancer cells to transiently transition between epithelial and mesenchymal states contributes to their metastatic potential. Therefore, driving tumor cells into a stable mesenchymal state, as opposed to complete tumor cell eradication, presents an opportunity to pharmacologically limit disease progression by promoting an asymptomatic state of dormancy. Here we compare a reversible model of epithelial-mesenchymal transition (EMT) induced by TGF-β to a stable mesenchymal phenotype induced by chronic exposure to the ErbB kinase inhibitor lapatinib. Only cells capable of returning to an epithelial phenotype resulted in skeletal metastasis. Gene expression analyses of the two mesenchymal states indicated similar transition expression profiles. A potently downregulated gene in both datasets was spleen tyrosine kinase (SYK). In contrast to this similar diminution in mRNA, kinome analyses using a peptide array and DNA-conjugated peptide substrates showed a robust increase in SYK activity upon TGF-β-induced EMT only. SYK was present in cytoplasmic RNA processing depots known as P-bodies formed during the onset of EMT, and SYK activity was required for autophagy-mediated clearance of P-bodies during mesenchymal-epithelial transition (MET). Genetic knockout of autophagy related 7 (ATG7) or pharmacological inhibition of SYK activity with fostamatib, a clinically approved inhibitor of SYK, prevented P-body clearance and MET, inhibiting metastatic tumor outgrowth. Overall, the current study suggests assessment of SYK activity as a biomarker for metastatic disease and the use of fostamatinib as a means to stabilize the latency of disseminated tumor cells

Approved and experimental small-molecule oncology kinase inhibitor drugs: a mid-2016 overview

Kinase inhibitor research is a comparatively recent branch of medicinal chemistry and pharmacology and the first small-molecule kinase inhibitor, imatinib, was approved for clinical use only 15 years ago. Since then, 33 more kinase inhibitor drugs have received regulatory approval for the treatment of a variety of cancers and the volume of reports on the discovery and development of kinase inhibitors has increased to an extent where it is now difficult—even for those working in the field—easily to keep an overview of the compounds that are being developed, as currently there are 231 such compounds, targeting 38 different protein and lipid kinases (not counting isoforms), in clinical use or under clinical investigation. The purpose of this review is thus to provide an overview of the biomedical rationales for the kinases being targeted on the one hand, and the design principles, as well as chemical, pharmacological, pharmaceutical, and toxicological kinase inhibitor properties, on the other hand. Two issues that are especially important in kinase inhibitor research, target selectivity and drug resistance, as well as the underlying structural concepts, are discussed in general terms and in the context of relevant kinases and their inhibitors

Syk and pTyr'd: Signaling through the B cell antigen receptor

AbstractThe B cell receptor (BCR) transduces antigen binding into alterations in the activity of intracellular signaling pathways through its ability to recruit and activate the cytoplasmic protein-tyrosine kinase Syk. The recruitment of Syk to the receptor, its activation and its subsequent interactions with downstream effectors are all regulated by its phosphorylation on tyrosine. This review discusses our current understanding of how this phosphorylation regulates the activity of Syk and its participation in signaling through the BCR