A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

Repression of SHP-1 expression by p53 leads to trkA tyrosine phosphorylation and suppression of breast cancer cell proliferation.

The nerve growth factor (NGF) receptor, trkA, the tumour suppressor p53 and the phosphatase SHP-1 are critical in cell proliferation and differentiation. SHP-1 is a trkA phosphatase that dephosphorylates trkA at tyrosines (Y) 674 and 675. p53 can induce trkA activation and tyrosine phosphorylation in the absence of NGF stimulation. In breast cancer tumours trkA expression is associated with increased patient survival. TrkA protein expression is higher in breast-cancer cell lines than in normal breast epithelia. In cell lines (but not in normal breast epithelia) trkA is functional and can be NGF-stimulated to promote cell proliferation. This study investigates the functional relationship between trkA, p53 and SHP-1 in breast-cancer, and reveals that in wild-type (wt) trkA expressing breast-cancer cells both endogenous wtp53, activated by therapeutic agents, and transfected wtp53 repress expression of SHP-1 through the proximal CCAAT sequence of the SHP-1-P1-promoter and the transcription factor NF-Y. In these cells trkA-Y674/Y675 phosphorylation is detected when SHP-1 protein levels decrease in a wtp53-dependent manner. Proliferation and cell-cycle assays, with cells expressing endogenous or transfected wt-trkA and a temperature-sensitive p53 grown at 32 °C (when p53 is in the wt configuration), show suppressed cell proliferation. Suppression is not detected when grown at 37 °C (when p53 is in the mutant configuration). A release from suppression is observed when these cells are transiently transfected with wt-SHP-1 and grown at 32 °C. Suppression is also detected when, as control, wt-trkA-expressing cells are transiently transfected with SHP-1-siRNA, but not when a dominant-negative (DN) mutant trkA is used to abolish wt-trkA activity. Importantly, suppression is not seen with control trkA-negative breast-cancer cells (expressing wtp53, wt-SHP-1 and undetectable trkA), transfected with Y674F/Y675F mutant-trkA. BrdU-incorporation experiments reveal lack of incorporation in cells expressing wt-trkA and wtp53, or wt-trkA and SHP-1-siRNA. However, BrdU is incorporated in the presence of Y674F/Y675F mutant trkA or DN mutant trkA. These results indicate that p53 repression of SHP-1 expression leads to trkA-Y674/Y675 phosphorylation and trkA-dependent suppression of breast-cancer cell proliferation. These data provide an explanation as to why high trkA levels are associated with favourable prognosisPeer reviewe