RSK2, also known as RPS6KA3 (ribosomal protein S6 kinase, 90 kDa, polypeptide 3), is a downstream kinase of the mitogen-activated protein kinase (MAPK) pathway, which is important in regulating survival, transcription, growth and proliferation. However, its biological role in mitotic progression is not well understood. In this study, we examined the potential involvement of RSK2 in the regulation of mitotic progression. Interestingly, depletion of RSK2, but not RSK1, caused the accumulation of mitotic cells. Time-lapse analysis revealed that mitotic duration, particularly the duration for metaphase-to-anaphase transition was prolonged in RSK2-depleted cells, suggesting activation of spindle assembly checkpoint (SAC). Indeed, more BubR1 (Bub1-related kinase) was present on metaphase plate kinetochores in RSK2-depleted cells, and depletion of BubR1 abolished the mitotic accumulation caused by RSK2 depletion, confirming BubR1-dependent SAC activation. Along with the shortening of inter-kinetochore distance, these data suggested that weakening of the tension across sister kinetochores by RSK2 depletion led to the activation of SAC. To test this, we analyzed the RSK2 effects on the stability of kinetochore–microtubule interactions, and found that RSK2-depleted cells formed less kinetochore–microtubule fibers. Moreover, RSK2 depletion resulted in the decrease of basal level of microtubule as well as an irregular distribution of mitotic spindles, which might lead to observed several mitotic progression defects such as increase in unaligned chromosomes, defects in chromosome congression and a decrease in pole-to-pole distance in these cells. Taken together, our data reveal that RSK2 affects mitotic progression by regulating the distribution, basal level and the stability of mitotic spindles.
Humans ; Kinetochores ; M Phase Cell Cycle Checkpoints ; Metaphase ; Microtubules ; Phosphotransferases ; Protein Kinases ; Ribosomal Protein S6 Kinases ; Ribosomal Protein S6 Kinases, 90-kDa* ; Siblings ; Spindle Apparatus*

PURPOSE: The 40S ribosomal protein S6 kinase-1 (S6K1) is a crucial downstream effector of the PI3K/AKT/mTOR pathway. S6K1 overexpression is found in 10% to 30% of breast cancers and is associated with aggressive disease and poor prognosis. Herein, we investigated the relationship between the expression of phosphorylated S6K1 (p-S6K1) and efficacy of lapatinib in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer. METHODS: We retrospectively analyzed the data of 36 patients with HER2-positive metastatic breast cancer treated with lapatinib between January 2010 and September 2014. The p-S6K1 expression status of the primary tumor was assessed via immunohistochemistry using a mouse monoclonal antibody. RESULTS: Fourteen of the 36 patients (38.9%) had p-S6K1-positive tumors. The median progression-free survival (PFS) of patients with p-S6K1-positive tumors was significantly longer than that of patients with p-S6K1-negative tumors (13.4 months vs. 7.1 months, p=0.025). In multivariate analysis, p-S6K1 positivity remained an independent, favorable predictive factor for PFS (hazard ratio, 0.32; 95% confidence interval, 0.11–0.97; p=0.044). CONCLUSION: The high expression of p-S6K1 was significantly associated with prolonged PFS, suggesting that p-S6K1 can be a potential biomarker for predicting the efficacy of lapatinib in patients with HER2-positive metastatic breast cancer.
Animals ; Breast Neoplasms* ; Breast* ; Disease-Free Survival ; Epidermal Growth Factor* ; Humans* ; Immunohistochemistry ; Mice ; Multivariate Analysis ; Prognosis ; Receptor, Epidermal Growth Factor* ; Retrospective Studies ; Ribosomal Protein S6 ; Ribosomal Protein S6 Kinases

PURPOSE: To evaluate the pathological prognostic factors related to local recurrence after radical surgery and adjuvant radiation therapy in advanced rectal cancer. MATERIALS AND METHODS: Fifty-four patients with advanced rectal cancer who were treated with radical surgery followed by adjuvant radiotherapy and chemotherapy between February 1993 and December 2001 were enrolled in this study. Among these patients, 14 patients experienced local recurrence. Tissue specimens of the patients were obtained to determine pathologic parameters such as histological grade, depth of invasion, venous invasion, lymphatic invasion, neural invasion and immunohistopathological analysis for expression of p53, Ki-67, c-erb, ezrin, c-met, phosphorylated S6 kinase, S100A4, and HIF-1 alpha. The correlation of these parameters with the tumor response to radiotherapy was statistically analyzed using the chi-square test, multivariate analysis, and the hierarchical clustering method. RESULTS: In univariate analysis, the histological tumor grade, venous invasion, invasion depth of the tumor and the over expression of c-met and HIF-1 alpha were accompanied with radioresistance that was found to be statistically significant. In multivariate analysis, venous invasion, invasion depth of tumor and over expression of c-met were also accompanied with radioresistance that was found to be statistically significant. By analysis with hierarchical clustering, the invasion depth of the tumor, and the over expression of c-met and HIF-1 alpha were factors found to be related to local recurrence. Whereas 71.4% of patients with local recurrence had 2 or more these factors, only 27.5% of patients without local recurrence had 2 or more of these factors. CONCLUSION: In advanced rectal cancer patients treated by radical surgery and adjuvant chemo-radiation therapy, the poor prognostic factors found to be related to local recurrence were HIF-1 alpha positive, c-met positive, and an invasion depth more than 5.5 mm. A prospective study is necessary to confirm whether these factors would be useful clinical parameters to measure and predict a radio-resistance group of patients.
Chemoradiotherapy* ; Drug Therapy ; Humans ; Multivariate Analysis ; Prospective Studies ; Radiotherapy ; Radiotherapy, Adjuvant ; Rectal Neoplasms* ; Recurrence* ; Ribosomal Protein S6 Kinases

It has been reported that most of Helicobacter pylori proteome components appear so crowded in the region of pH 4.5~8.0 that a lot of them were inseparable in 2-DE using the broad range IPG strip. Therefore, inseparable protein spots in 2-DE profiles have to be apart from each other for improving the protein identification. Here, we attempt to examine the usability of the narrow range IPG strips for separating close spots in the broad range IPG strip at proteomic analysis of H. pylori. The whole cell proteins of H. pylori strain 26695 were separated by narrow range IPG strips (pI 3.9~5.1, 4.7~5.9, 5.5~6.7, and 6.3~8.3, respectively), followed by SDS-PAGE, and visualized by silver staining, showing that the distances between spots were widened and the total number of detectable spots was increased. Resolved protein spots were identified by the peptide fingerprinting using MALDI-TOF-MS. As a result, 87 expressed proteins were identified by the peptide fingerprinting. Of them, 23 proteins, including hydrogenase expression/formation protein, purine-binding chemotaxis protein, and ribosomal protein S6, have not been reported in the previous proteome studies of H. pylori. Thus, these results demonstrate that the high complexity proteome components could be effectively separated using the narrow range IPG strips, which might be helpful to strengthen the contents of the master protein map of the H. pylori reference strain.
Chemotaxis ; Electrophoresis, Polyacrylamide Gel ; Helicobacter pylori* ; Helicobacter* ; Hydrogen-Ion Concentration ; Hydrogenase ; Peptide Mapping ; Proteome ; Proteomics ; Ribosomal Protein S6 ; Silver Staining

OBJECTIVETo study the role of mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70 S6K) in periodontal tissues remodeling during orthodontic tooth movement.

METHODSTwenty-four rabbits were chosen to establish rabbit models for the study. The right maxillary teeth of each animal treated by orthodontics were the test sides, and the untreated left teeth were the control sides. The animals were sacrificed at 3, 5, 7, 14 d, respectively. The prepared tissue specimens were processed for the study of the change in the histologic morphology with HE staining and the expression of p70 S6K in periodontal tissues by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot techniques.

RESULTSIn the test side, the tranecula of bone became porous and cells arrayed disorderly, resorption of alveolar bone was observed in histological study. There were many holes in the alveolar bone, osteoblasts were occasionally observed. RT-PCR showed that the expression of p70 S6K mRNA dramatically changed at 3 d. The expression of p70 S6K mRNA in treating periodontal tissues was higher than the control side, especially at 7 d, and then decreased. Compared with the control side, there was significant difference in statistical analysis (P<0.01). The expression of Western blot was same as the RT-PCR.

CONCLUSIONHigher expression of mTOR and p70 S6K during orthodontic tooth movement especially plays an important role in the process of periodontium remodeling.

Animals ; Osteoblasts ; Periodontal Ligament ; Rabbits ; Ribosomal Protein S6 Kinases, 70-kDa ; Sirolimus ; TOR Serine-Threonine Kinases ; Tooth Movement Techniques

p70 ribosomal protein S6 kinase (p70S6K), an important member of AGC family, is a kind of multifunctional Ser/Thr kinases, which plays an important role in mTOR signaling cascade. The p70 ribosomal protein S6 kinase is closely associated with diverse cellular processes such as protein synthesis, mRNA processing, glucose homeostasis, cell growth and apoptosis. Recent studies have highlighted the important role of S6K in cancer, which arose interests of scientific researchers for the design and discovery of anti-cancer agents. Herein, the mechanisms of S6K and available inhibitors are reviewed.
Antineoplastic Agents ; Humans ; Protein Kinase Inhibitors ; chemistry ; Ribosomal Protein S6 Kinases, 70-kDa ; antagonists & inhibitors ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases

Ribosomal S6 kinase is a family of serine/threonine protein kinases involved in the regulation of cell viability. There are two subfamilies of ribosomal s6 kinase, (p90rsk, p70rsk). Especially, p90rsk is known to be an important downstream kinase of p44/42 MAPK. We investigated the role of p90rsk on ethanol-induced cell proliferation of HepG2 cells. HepG2 cells were treated with 10~50 mM of ethanol with or without ERK and p90rsk inhibitors. Cell viability was measured by MTT assay. The expression of pERK1, NHE1 was measured by Western blots. The phosphorylation of p90rsk was measured by ELISA kits. The expression of Bcl-2 was measured by qRT-PCR. When the cells were treated with 10~30 mM of ethanol for 24 hour, it showed significant increase in cell viability versus control group. Besides, 10~30 mM of ethanol induced increased expression of pERK1, p-p90rsk, NHE1 and Bcl-2. Moreover treatment of p90rsk inhibitor attenuated the ethanol-induced increase in cell viability and NHE1 and Bcl-2 expression. In summary, these results suggest that p90rsk, a downstream kinase of ERK, plays a stimulatory role on ethanol-induced hepatocellular carcinoma progression by activating anti-apoptotic factor Bcl-2 and NHE1 known to regulate cell survival.
Blotting, Western ; Carcinoma, Hepatocellular ; Cell Proliferation* ; Cell Survival ; Enzyme-Linked Immunosorbent Assay ; Ethanol ; Hep G2 Cells* ; Humans ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; Ribosomal Protein S6 Kinases

We attempted to study the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the cascade of phosphorylation of ribosomal protein S6 during differentiation of leukemic cells (HL-60, THP-1, and RWLeu-4). Neither activation nor inhibition of colony stimulating factor-1 (CSF-1) receptor's PTK activity with CSF-1 or genistein respectively affected the phosphorylation of S6. However, vanadate which is a protein tyrosine phosphatase (PTP) inhibitor showed enhancement of S6 phosphorylation. Dimethylsulfoxide which does not affect either PTK or PKC demonstrated no change in S6 phosphorylation. PKC activation by acute 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment induced monocytic differentiation and S6 phosphorylation. Surprisingly, the more prominent phosphorylation of S6 protein was observed in PKC-depleted cells by prolonged TPA treatment. Our results suggest that PTK/PTP play a lesser role in S6 phosphorylation of HL-60 cells than PKC does. In addition, two different mechanisms seem to be involved in TPA-induced S6 phosphorylation during HL-60 differentiation: PKC activation by acute TPA treatment and PKC depletion which may lead to the synthesis of some endogenous protein responsible for the differentiation by chronic TPA treatment.
Cell Differentiation ; Humans ; Leukemia/*metabolism/pathology ; Macrophage Colony-Stimulating Factor/pharmacology ; Phosphorylation ; Protein Kinase C/physiology ; Protein-Tyrosine Kinases/physiology ; Ribosomal Protein S6 ; Ribosomal Proteins/*metabolism ; Tetradecanoylphorbol Acetate/pharmacology ; Tumor Cells, Cultured

Obesity is a prevalent metabolic disorder, which seriously affects human health and has become the world's public health problem. Kinase S6K1, an important downstream effector of mammalian target of rapamycin (mTOR), influences specific pathological responses, including obesity, type 2 diabetes and cancer. Presently, S6K1 has become an attractive therapeutic target in the treatment of these disorders. Here, the functions of kinase S6K1, its molecular regulation mechanisms, related pathogenesis of disease and relevant small molecular inhibitors are reviewed. Finally, the prospect of research toward S6K1 is expected as well.
Animals ; Diabetes Mellitus, Type 2 ; Humans ; Neoplasms ; Obesity ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism

OBJECTIVETo investigate the effect of rapamycin on the proliferation of rat valvular interstitial cells in primary culture.

METHODSThe interstitial cells isolated from rat aortic valves were cultured and treated with rapamycin, and the cell growth and cell cycle changes were analyzed using MTT assay and flow cytometry, respectively. RT-PCR was used to detect mRNA expression levels of S6 and P70S6K in cells, and the protein expressions level of S6, P70S6K, P-S6, and P-P70S6K were detected using Western blotting.

RESULTSRat aortic valvular interstitial cells was isolated successfully. The rapamycin-treated cells showed a suppressed proliferative activity (P<0.05), but the cell cycle distribution remained unaffected. Rapamycin treatment resulted in significantly decreased S6 and P70S6K protein phosphorylation level in the cells (P<0.05).

CONCLUSIONThe mechanism by which rapamycin inhibits the proliferation of valvular interstitial cells probably involves suppression of mTOR to lower S6 and P70S6K phosphorylation level but not direct regulation of the cell cycle.

Animals ; Blotting, Western ; Cell Cycle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Heart Valves ; cytology ; Phosphorylation ; Rats ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Sirolimus ; pharmacology