A surface-activated chemical ionization approach allows quantitative phosphorylation analysis of the cyclin-dependent kinase inhibitor Sic1 phosphorylated on Ser201.

RATIONALE: Quantitative phosphoproteomics represents a front line for functional proteomics and hence for systems biology. Here we present a new application of the surface-activated chemical ionization (SACI) technology for quantitative phosphoproteomics analysis. The main advantages of SACI-MS technology are high sensitivity, quantitative accuracy and matrix effect reduction, which allow quantitative estimations. METHODS: A SACI-MS approach was used to investigate the quantitative in vivo phosphorylation of the cyclin-dependent kinase inhibitor Sic1, a low-abundance protein of Saccharomyces cerevisiae, which is phosphorylated on Ser201 by casein kinase 2 (CK2) and compared its phosphorylation status in cells growing in two different carbon sources (glucose or ethanol). RESULTS: Our relative quantification indicated that the Sic1-Ser201 phosphorylation level is about 2-fold higher in ethanol- than in glucose-growing cells, proportional to the Sic1 protein level. This finding is coherent with results of western blot analysis using anti-phospho-Ser201-specific antibody, validating the results obtained with this new SACI approach. CONCLUSIONS: The findings presented in this paper indicate that the innovative LC/SACI-MS method, coupled with immunoprecipitation, is a powerful device to obtain quantitative information on the phosphorylation state of low abundance proteins.

Functions, regulation and cellular localization of plant cyclin-dependent kinase inhibitors.

The cell cycle is regulated by the cyclin-dependent kinase (CDK), and CDK inhibitors can bind to CDKs and inhibit their activities. This review examines plant CDK inhibitors, with particular emphasis on their molecular and cellular functions, regulation and cellular localization. In plants, a family of ICK/KRP CDK inhibitors represented by ICK1 is known and another type of CDK inhibitor represented by the SIMESE (SIM) has recently been reported. Considerable understanding has been gained with the ICK/KRP CDK inhibitors. These plant CDK inhibitors share only limited sequence similarity in the C-terminal region with the KIP/CIP family of mammalian CDK inhibitors. The ICK/KRP CDK inhibitors thus provide good tools to understand the basic machinery as well as the unique aspects of the plant cell cycle. The ICK/KRP CDK inhibitors interact with D-type cyclins or A-type CDKs or both. Several functional regions and motifs have been identified in ICK1 for CDK inhibition, nuclear localization and protein instability. Clear evidence shows that ICK/KRP proteins are important for the cell cycle and endoreduplication. Preliminary evidence suggests that they may also be involved in cell differentiation and cell death. Results so far show that plant CDK inhibitors are exclusively localized in the nucleus. The molecular sequences regulating the localization and functional significance will be discussed.

Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro.

The objective of this study was to investigate the effects of selenium (Se) on in vitro proliferation, apoptosis and testosterone production of sheep Leydig cells and its underlying mechanism. Leydig cells were collected from 8-month-old sheep and divided into four treatment groups (0, 2.0, 4.0 and 8.0 µmol/L Se). After treatment with Se for 48 h, the MTT and flow cytometric assay were used to detect cell proliferation and apoptosis. Testosterone level in the culture medium was determined by ELISA. The mRNA expression and protein abundance of cell cycle, apoptosis and testosterone synthesis-related genes were detected using real-time PCR and western blot analysis. The results showed that the highest percentage of live and apoptotic cells was obtained in the 2.0 and 8.0 µmol/L group, respectively. In the Se treatment groups, the proliferation rate of Leydig cells and the expression of cell cycle-related genes were decreased with the increasing Se supplementation in the culture medium. The percentage of apoptotic cells was increased with the increasing Se level, which was consistent with the expression of pro-apoptosis genes. The highest GSH-Px activity and lowest ROS content were also observed in the 2.0 µmol/L group. Appropriate Se level (2.0 µmol/L) can significantly increase the expression of p-ERK1/2, StAR and 3ß-HSD, and improve the testosterone synthesis. Compared with the control group, PD0325901 could significantly inhibit the production of testosterone and the protein abundance of p-ERK1/2, StAR and 3ß-HSD. Se treatment can mitigate the inhibition effect of PD0325901 and the testosterone secretion between the 2.0 µmol/L and control group was not significantly different. These results demonstrate that Se can affect the proliferation and apoptosis of Leydig cells by regulating cellular oxidative stress and the expressions of cell cycle and apoptosis-related genes. Se can also enhance the testosterone production of Leydig cells by activating the ERK signaling pathway and the expression of its downstream genes (StAR and 3ß-HSD), which could be closely related to the regulating roles of Se in male fertility and spermatogenesis.

The tumor suppressor CDKN3 controls mitosis.

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2(pThr-161) at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKß phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.

Cell cycle regulatory proteins in the liver in murine Trypanosoma cruzi infection.

The liver is an important target of Trypanosoma cruzi infection. Infection of CD-1 mice with T. cruzi (Brazil strain) resulted in parasitism of the liver, primarily in sinusoidal and Kupffer cells. Immunoblot analysis revealed activation of extra cellular signal-regulated kinase (ERK) during the acute and subacute period of infection, but p38 mitogen activated kinase (MAPK) and JNK were not activated. The activity of important cell cycle regulatory genes was also examined in the liver following infection. There was increased expression of cyclin D1, cyclin E and cyclin A as well as proliferating cell nuclear antigen (PCNA) at 45, 60 and 215 days post infection. In addition, the levels of the cyclin-dependent kinase inhibitors p27(KIP1), p21(WAF1) and the tumor suppressor p53 were increased in the livers obtained from infected mice. Quantitative PCR revealed increased abundance of mRNA for cyclins A, D1 and E. Interestingly, cyclin A and E are ordinarily not found in the adult liver. Thus infection caused a reversion to a fetal/neonatal phenotype. These data provide a molecular basis for cell proliferation in the liver following T. cruzi infection.