A TOR (target of rapamycin) and nutritional phosphoproteome of fission yeast reveals novel targets in networks conserved in humans

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe . Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPK α ( ssp2::ura4 + ), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.

Comparative analysis of two murine CDC25B isoforms

CDC25B phosphatase plays a pivotal role in the cell cycle process by dephosphorylating and activating the CDC2 kinase of maturation-promoting factor (MPF). In mice, two transcripts of Cdc25B are generated by the alternative splicing of one gene. We compared the properties of these two forms of CDC25B. When the expression pattern of Cdc25B was examined using RT-PCR, both forms were detected in almost all mouse tissues tested. The expression of two forms of the CDC25B protein in various mouse tissues was confirmed using Western blotting with generated isoform specific antibodies. CDC25B1 tends to accumulate more in the cytosol than CDC25B2 does, and they have different binding capacity for 14-3-3 proteins. CDC25B1 was more effective in dephosphorylating in vitro substrate para-nitrophenyl phosphate and showed higher activity in the modified histone H1 kinase assay than CDC25B2. These results suggest that the two forms of CDC25B play different roles in cell cycle regulation.

Leaf Extracts ofCalocedrus formosana(Florin) Induce G2/M Cell Cycle Arrest and Apoptosis in Human Bladder Cancer Cells

Calocedrus formosana(Florin) bark acetone/ethylacetate extracts are known to exert an antitumor effect on some human cancer cell lines, but the mechanism is yet to be defined. The aim of this study was to determine the effects of Florin leaf methanol extracts on the growth and apoptosis of human bladder cancer cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that the growth of these bladder cancer cells was potently inhibited by the Florin leaf extracts. The cell cycle of these extract-treated cells (TCCSUP cells) was arrested at the G2/M phase as determined by flow cytometry. Western blot analysis revealed the increases of cyclin B1 and Cdc2 kinase levels, alone with the decrease of phosphorylated Cdc2 kinase, after treating these cells with the extracts. An immunofluorescence assessment ofβ-tubulin showed decreased levels of polymerized tubulin in treated cells. However, the proteolytic cleavage of poly ADP-ribose polymerase and the activation of caspase-3/-8/-9 were all increased upon treatments of extracts. The concurrent increase of Bax and decrease of Bcl-2 levels indicated that the extracts could induce apoptosis in these treated cells. Taken together, these results suggest that the Florin leaf extracts may be an effective antibladder cancer agent.

Cdc2 and Mos Regulate Emi2 Stability to Promote the Meiosis I–Meiosis II Transition

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca2+/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.