Importance of Validating Antibodies and Small Compound Inhibitors Using Genetic Knockout Studies-T Cell Receptor-Induced CYLD Phosphorylation by IKKε/TBK1 as a Case Study

Frontiers in Cell and Developmental Biology
Marie LorkRudi Beyaert

Abstract

CYLD is a deubiquitinating enzyme that plays a crucial role in immunity and inflammation as a negative regulator of NF-κB transcription factor and JNK kinase signaling. Defects in either of these pathways contribute to the progression of numerous inflammatory and autoimmune disorders. Therefore, we set out to unravel molecular mechanisms that control CYLD activity in the context of T cell receptor (TCR) signaling. More specifically, we focused on CYLD phosphorylation at Ser418, which can be detected upon immunoblotting of cell extracts with phospho(Ser418)-CYLD specific antibodies. Jurkat T cells stimulated with either anti-CD3/anti-CD28 or PMA/Ionomycin (to mimic TCR signaling) were used as a model system. The role of specific kinases was analyzed using pharmacological as well as genetic approaches. Our initial data indicated that CYLD is directly phosphorylated by the noncanonical IκB kinases (IKKs) IKKε and TANK Binding Kinase 1 (TBK1) at Ser418 upon TCR stimulation. Treatment with MRT67307, a small compound inhibitor for IKKε and TBK1, inhibited TCR-induced CYLD phosphorylation. However, the phospho(Ser418)-CYLD immunoreactive band was still present in CRISPR/Cas9 generated IKKε/TBK1 double knockout cell lines, where it cou...Continue Reading

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Citations

Sep 19, 2018·Cells·Joel K DurandAlbert S Baldwin
Dec 6, 2018·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Laura MeloniRudi Beyaert
Mar 19, 2020·Brain : a Journal of Neurology·Carol Dobson-StoneJohn B Kwok
Jul 8, 2020·International Journal of Molecular Sciences·Moritz MöllerEllen Niederberger

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Methods Mentioned

BETA
immunoprecipitation
protein assay
transfection
PMA
deubiquitination
genetic knockout

Software Mentioned

Tracking of by DEcomposition ( TIDE )

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