MOK

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease affecting motor neurons and characterized by microglia-mediated neurotoxic inflammation whose underlying mechanisms remain incompletely understood. In this work, we reveal that MAPK/MAK/MRK overlapping kinase (MOK), with an unknown physiological substrate, displays an immune function by controlling inflammatory and type-I interferon (IFN) responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as an effector protein regulated by MOK, by promoting Ser⁴⁹²-phospho-Brd4 levels. We further demonstrate that MOK regulates Brd4 functions by supporting its binding to cytokine gene promoters, therefore enabling innate immune responses. Remarkably, we show that MOK levels are increased in the ALS spinal cord, particularly in microglial cells, and that administration of a chemical MOK inhibitor to ALS model mice can modulate Ser⁴⁹²-phospho-Brd4 levels, suppress microglial activation, and modify the disease course, indicating a pathophysiological role of MOK kinase in ALS and neuroinflammation.

MAPK/MAK/MRK Overlapping Kinase (MOK) belongs to MAP kinase superfamily, which plays an important role in regulating cell growth, division, and differentiation. Caspase-3, as the final executor of apoptosis, has an important position in the caspase-mediated apoptotic signaling pathway. The full-length cDNA of MOK and caspase-3 were cloned from Cristaria plicata (designated CpMOK and CpCaspase-3). The CpMOK gene was sequence with a full-length of 1413 bp, encoding a total of 470 amino acids, and containing an S_TKc structural domain. CpCaspase-3 has a sequence of 2425 bp, encoding 322 amino acids, containing a CASc domain. Real-time fluorescence quantitative PCR analysis showed that CpMOK and CpCaspase-3 distributed in various tissues of C. plicata, and the highest expression of CpMOK and CpCaspase-3 mRNA was in hepatopancreas. The expression of CpMOK was significantly changed in hepatopancreas, gills, and kidneys by the construction of wound model as well as stimulation of LPS, PGN, Poly I: C and Aeromonas hydrophila. Subcellular localization experiments confirmed that CpMOK was localized in the nucleus. Furthermore, the double-stranded RNA (dsRNA) of CpMOK was constructed for interference experiment, and the results showed that the mRNA expression of apoptotic gene signals caspase-1, caspase-3, caspase-7, caspase-8, and caspase-9 were increased. The expression of caspase-1, -3, -7, -9, cytochrome C (Cyt-c) and tumor necrosis factor-α (TNF-α) was detected by ELISA. Fluorescent staining of apoptotic cells using the Tunnel method revealed an increase in the number of apoptotic cells after interference. These results suggested that CpMOK knockdown could induce caspase-mediated apoptosis in C. plicata, and the phosphorylation of the kinase was disrupted during the process.

Chordoma is a slow-growing but malignant subtype of bone sarcoma with relatively high recurrence rates and high resistance to chemotherapy. It is urgent to understand the underlying regulatory networks to determine more effective potential targets. Phosphorylative regulation is currently regarded as playing a significant role in tumorigenesis, and the use of tyrosine kinase inhibitors in clinical practice has yielded new promise for the treatment of a variety of sarcoma types.

We performed comprehensive proteomic and phosphoproteomic analyses of chordoma using four-dimensional label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis. The potential aberrantly expressed kinases and their functions were validated using western blotting and CCK-8 assays.

Compared with paired normal muscle tissues, 1,139 differentially expressed proteins (DEPs) and 776 differentially phosphorylated proteins (DPPs) were identified in chordoma tumor tissues. The developmentally significant Wnt-signaling pathway and oxidative phosphorylation were aberrant in chordoma. Moreover, we predicted three kinases (AURA, CDK9, and MOK) with elevated activity by kinase-pathway network analysis (KiPNA) and verified their increased expression levels. The knockdown of these kinases markedly suppressed chordoma cell growth, and this was also the case for cells treated with the CDK9 inhibitor AZD4573. We additionally examined 208 proteins whose expression and phosphorylation levels were synergetically altered.

We herein depicted the collective protein profiles of chordomas, providing insight into chordomagenesis and the potential development of new therapeutic targets.