1C). (+/?) for ZPR9, NIH 3T3 cells with inducible knockdown of ZPR9, and CRISPR/Cas9-mediated ZPR9 knockout cells. Furthermore, high-fat diet (HFD)-fed mice displayed reduced MPK38 kinase activity and ZPR9 expression compared to AGI-5198 (IDH-C35) that in mice on control chow, suggesting that ZPR9 acts as a physiological AGI-5198 (IDH-C35) activator of MPK38 that may participate in obesity. Emerging evidence has implicated ASK1/TGF\/Smad3 signaling in the pathogenesis of obesity-associated metabolic diseases. For instance, ASK1 signaling has been shown to associate with TGF\ signaling and to contribute to the improvement of glucose and lipid metabolism in genetically and diet-induced obese mice1. TGF\ signaling has been shown to be involved in numerous metabolic processes, including systemic glucose and lipid metabolism, pancreatic -cell function, adipocyte differentiation, AGI-5198 (IDH-C35) adipocytokine secretion and inflammation2. In addition, evidence of interplay between p53 and mechanistic target of rapamycin (mTOR) signaling, glucose and lipid metabolism, or mitochondrial maintenance suggested that p53 also plays a crucial role in the regulation of cellular metabolic homeostasis3. MPK38, otherwise known as maternal embryonic leucine zipper kinase (MELK), was an AMPK\related serine-threonine kinase that was highly conserved across different species. It regulated a variety of cellular processes, including the cell cycle, cell proliferation, spliceosome assembly, carcinogenesis, stem cell AGI-5198 (IDH-C35) self\renewal, apoptosis, and numerous signal transduction pathways4,5,6,7,8,9,10,11,12,13. MPK38 was activated by different stimuli, including H2O2, tumor necrosis factor- (TNF-), thapsigargin, ionomycin, TGF-1, 5-fluorouracil (5FU), and doxorubicin (Dox), that trigger ASK1, TGF-, and p53 signaling pathways5,14,15,16. The intracellular signaling proteins ASK1, Smads, and p53 were recently found to interact with MPK38 was validated in ZPR9 (T252A) knockin 3T3-L1 cell lines generated by the CRISPR/Cas9 system. MPK38 kinase reactions including OTSSP167, a MPK38-specific inhibitor24, showed clearly that both endogenous AGI-5198 (IDH-C35) and recombinant MPK38 proteins directly phosphorylate ZPR9 at Thr252 (Fig. 1B). In addition, a stoichiometric analysis of ZPR9 phosphorylation by MPK38 supported that MPK38 phosphorylates only one residue (Thr252) in ZPR9 (Fig. 1C). We then measured the kinase activity of MPK38 in the presence or absence of ZPR9 using kinase assays with recombinant MPK38 proteins (Fig. 2A). The coexpression of ZPR9 markedly increased the kinase activity of MPK38, indicating that ZPR9 positively regulates MPK38 activity. To investigate whether the ability of ZPR9 to stimulate MPK38 kinase activity requires both direct conversation with and phosphorylation by MPK38, we also screened the ZPR9 T252A mutant in kinase assays. The coexpression of the T252A mutant had no effect on MPK38 kinase activity compared to that of the control, which contained wild\type MPK38 recombinant protein alone. Consistently, the kinase activity of MPK38 was considerably decreased U2AF1 in ZPR9 knockout and (T252A) knockin 3T3-L1 cells compared to control wild-type 3T3-L1 cells (Fig. 2B and Supplementary Fig. S1). The treatment of wild-type 3T3-L1 cells with OTSSP167 also displayed no MK38 kinase activity. These results support again a central role for ZPR9 phosphorylation at Thr252 by MPK38 in the ZPR9-mediated stimulation of MPK38 kinase activity. On the contrary, the phosphomimetic mutation T252D displayed high levels of MPK38 kinase activity comparable to that of the wild-type ZPR9 (Fig. 2C), providing additional validation of the requirement of ZPR9 phosphorylation at Thr252 in the stimulation of MPK38 kinase activity. Together, these results suggest a critical role for both a redox-dependent conversation with and phosphorylation by MPK38 in the ZPR9-mediated stimulation of MPK38 kinase activity. Open in a separate window Physique 1 ZPR9 phosphorylation at Thr252 by MPK38.(A) To identify.
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