Abstract
BackgroundThe IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKβ, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied.MethodsMolecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry.ResultsWe show that IKKα and IKKβ bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3β, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers.ConclusionsOur data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.
Highlights
The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKβ, is the main activator of the inflammatory transcription factor nuclear factor κB (NF-κB), which is constitutively active in many cancers
We propose a model in which IKKα phosphorylates CHUK (IKKα)/MYC (c-Myc) at serine-67 and serine-71 in the nucleus, leading to subsequent inhibition of Glycogen synthase kinase 3β (GSK3β) mediated phosphorylation, which is important for degradation of c-Myc, stabilizing c-Myc and enhancing its transcriptional activation, thereby increasing proliferation and inhibiting apoptosis
IKKα directly interacts with c-Myc in the nucleus IKKα and IKKβ exhibit high similarities in their structure both containing a NH2-terminal protein kinase domain and leucine zipper (LZ) and helix-loop-helix (HLH) carboxy-terminal motifs, while showing several differences in their molecular functions and kinase activities [7]
Summary
The IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKβ, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. Many different stimuli or cellular stress conditions, such as inflammatory cytokines, or components of pathogens, cause an activation of the IKK complex, leading to phosphorylation and degradation of NF-κB inhibiting proteins or domains This releases the NF-κB dimer and results in a shift of its steady state localization into the nucleus, where it activates a high number of target genes, depending on the cell type and chromatin accessibility [3,4,5,6]. IKKα and IKKβ are biochemically and structurally related and act in a concerted manner within the IKKcomplex, they do not have identical physiological roles and differ in specific molecular functions, which they can exert outside the IKK-complex This is exemplified by the fact that they can phosphorylate IκB molecules, and other substrates with distinct specificities thereby regulating a multitude of biological functions [7]. This study used an IKK-inhibitor, which latter turned out to be very unspecific [17] and employed overexpression of IKKα or IKKβ, while we tried to elucidate the functional links between IKKs and c-Myc using CRISPR/Cas9-mediated gene knockout (KO) and further functional assays
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