Abstract
BackgroundDietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway.MethodsThe activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases.ResultsITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected.ConclusionThese results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins.
Highlights
Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle
ITCs inhibit MAPK/extracellular signal regulating kinase (ERK) kinase 1 (MEKK1), but not the related protein kinase Apoptosis signal regulating kinase 1 (ASK1), in intact cells To determine the effect of isothiocyanates on MEKK1 activity, we expressed a full-length MEKK1 protein fused to a chitin binding domain (CBD) tag to allow purification of the protein by binding to chitin beads
The cells were treated with increasing concentrations of phenethyl isothiocyanate (PEITC), and each kinase was purified by affinity chromatography using chitin beads followed by in vitro kinase assay using a bacterially-expressed inactive mutant of SAPK/ERK kinase (SEK) (SEK-KR) as substrate
Summary
Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. The MEKK1 protein kinase is a critical upstream mediator in signaling pathways that control the response of cells to stress stimuli. We recently demonstrated that MEKK1 is inhibited by oxidative stress stimuli through a mechanism involving direct glutathionylation of a specific cysteine residue in the ATP binding pocket [10]. This thiol modification is reversible by reducing agents, including glutathione, in vitro, and likely represents a reversible means of inhibiting the kinase activity within the cell during the response to oxidative insult
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.