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Mps1 is a protein kinase that regulates normal mitotic progression and the spindle checkpoint in response to spindle damage. The levels of Mps1 are relatively low in cells during interphase but elevated in mitosis or upon activation of the spindle checkpoint, although the dynamic range of Mps1 expression and the Mps1 catalytic mechanism have not been carefully characterized. Our recent structural studies of the Mps1 kinase domain revealed that the carboxyl-terminal tail region of Mps1 is unstructured, raising the question of whether this region has any functional role in Mps1 catalysis. Here we first determined the cellular abundance of Mps1 during cell cycle progression and found that Mps1 levels vary between 60,000 per cell in early G 1 and 110,000 per cell during mitosis. We studied phosphorylation of a number of Mps1 substrates in vitro and in culture cells. Unexpectedly, we found that the unstructured carboxyl-terminal region of Mps1 plays an essential role in substrate recruitment. Kinetics studies using the purified recombinant wild type and mutant kinases indicate that the carboxyl-terminal tail is largely dispensable for autophosphorylation of Mps1 but critical for trans-phosphorylation of substrates in vitro and in cultured cells. Mps1 mutant without the unstructured tail region is defective in mediating spindle assembly checkpoint activation. Our results underscore the importance of the unstructured tail region of Mps1 in kinase activation.

BackgroundAmong the various ginseng strains, Shizhu ginseng is endemic to China, mainly distributed in Kuandian Manchu Autonomous County (Liaoning Province, China); however, not much is known about the compounds (especially saponins) in Shizhu ginseng.MethodsA rapid, sensitive, and reliable ultra-high performance liquid chromatography coupled with MS/MS (UHPLC–MS/MS) method was developed to separate and identify saponins in Shizhu ginseng.ResultsThe separation was carried out on a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phase under a gradient elution at 40°C. The detection was performed on a Micromass Quattro Micro API mass spectrometer equipped with electrospray ionization source in both positive and negative modes. Under the optimized conditions, a total of 31 saponins were identified or tentatively characterized by comparing retention time and MS data with related literatures and reference substances.ConclusionThe developed UHPLC–MS/MS method was suitable for identifying and characterizing the chemical constituents in Shizhu ginseng, which provided a helpful chemical basis for further research on Shizhu ginseng.

Two new phenylpropanoid glycosides, smilasides M and N, together with the known compound 2',6'-diacetyl-3,6-diferuloylsucrose, were isolated and characterized from the roots and rhizomes of Smilax riparia A. DC. The structures of the new compounds were elucidated as 2',6'-diacetyl-3-Z-feruloyl-6-feruloylsucrose (1) and 2',6'-diacetyl-3-feruloyl-6-Z-feruloylsucrose (2) on the basis of extensive analysis of HR-ESI-MS, UV, IR, and 1D and 2D NMR spectroscopic data.