Cyclin-dependent Kinase Substrate Research Articles

CDK activity at the centrosome regulates the cell cycle

In human cells and yeast, an intact "hydrophobic patch" substrate docking site is needed for mitotic cyclin centrosomal localization. A hydrophobic patch mutant (HPM) of the fission yeast mitotic cyclin Cdc13 cannot enter mitosis, but whether this is due to defective centrosomal localization or defective cyclin-substrate docking more widely is unknown. Here, we show that artificially restoring Cdc13-HPM centrosomal localization promotes mitotic entry and increases CDK (cyclin-dependent kinase) substrate phosphorylation at the centrosome and in the cytoplasm. We also show that the S-phase B-cyclin hydrophobic patch is required for centrosomal localization but not for S phase. We propose that the hydrophobic patch is essential for mitosis due to its requirement for the local concentration of cyclin-CDK with CDK substrates and regulators at the centrosome. Our findings emphasize the central importance of the centrosome as a hub coordinating cell-cycle control and explain why the cyclin hydrophobic patch is essential for mitosis.

Live-cell imaging defines a threshold in CDK activity at the G2/M transition

Cyclin-dependent kinase (CDK) determines the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK and phosphorylation patterns of CDK substrates at the population level, it remains elusive how CDK regulators coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by developing a CDK activity biosensor, Eevee-spCDK. We find that although CDK activity does not necessarily correlate with cyclin levels, it converges to the same level around mitotic onset in several mutant backgrounds, including pom1Δ cells and wee1 or cdc25 overexpressing cells. These data provide direct evidence that cells enter the M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.

LAFEM: A Scoring Model to Evaluate Functional Landscape of Lysine Acetylome

Protein lysine acetylation is a critical post-translational modification involved in a wide range of biological processes. To date, about 20,000 acetylation sites of Homo sapiens were identified through mass spectrometry–based proteomic technology, but more than 95% of them have unclear functional annotations because of the lack of existing prioritization strategy to assess the functional importance of the acetylation sites on large scale. Hence, we established a lysine acetylation functional evaluating model (LAFEM) by considering eight critical features surrounding lysine acetylation site to high-throughput estimate the functional importance of given acetylation sites. This was achieved by selecting one of the random forest models with the best performance in 10-fold cross-validation on undersampled training dataset. The global analysis demonstrated that the molecular environment of acetylation sites with high acetylation functional scores (AFSs) mainly had the features of larger solvent-accessible surface area, stronger hydrogen bonding–donating abilities, near motif and domain, higher homology, and disordered degree. Importantly, LAFEM performed well in validation dataset and acetylome, showing good accuracy to screen out fitness directly relevant acetylation sites and assisting to explain the core reason for the difference between biological models from the perspective of acetylome. We further used cellular experiments to confirm that, in nuclear casein kinase and cyclin-dependent kinase substrate 1, acetyl-K35 with higher AFS was more important than acetyl-K9 with lower AFS in the proliferation of A549 cells. LAFEM provides a prioritization strategy to large scale discover the fitness directly relevant acetylation sites, which constitutes an unprecedented resource for better understanding of functional acetylome.

Distinct states of nucleolar stress induced by anticancer drugs

Ribosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed ‘nucleolar normality score’ was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I– recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development.

Distinct states of nucleolar stress induced by anticancer drugs

Ribosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed ‘nucleolar normality score’ was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I– recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development.

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing cyclin-dependent kinases (CDKs). However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

The ATM-E6AP-MASTL axis mediates DNA damage checkpoint recovery.

Checkpoint activation after DNA damage causes a transient cell cycle arrest by suppressing cyclin-dependent kinases (CDKs). However, it remains largely elusive how cell cycle recovery is initiated after DNA damage. In this study, we discovered the upregulated protein level of MASTL kinase hours after DNA damage. MASTL promotes cell cycle progression by preventing PP2A/B55-catalyzed dephosphorylation of CDK substrates. DNA damage-induced MASTL upregulation was caused by decreased protein degradation, and was unique among mitotic kinases. We identified E6AP as the E3 ubiquitin ligase that mediated MASTL degradation. MASTL degradation was inhibited upon DNA damage as a result of the dissociation of E6AP from MASTL. E6AP depletion reduced DNA damage signaling, and promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner. Furthermore, we found that E6AP was phosphorylated at Ser-218 by ATM after DNA damage and that this phosphorylation was required for its dissociation from MASTL, the stabilization of MASTL, and the timely recovery of cell cycle progression. Together, our data revealed that ATM/ATR-dependent signaling, while activating the DNA damage checkpoint, also initiates cell cycle recovery from the arrest. Consequently, this results in a timer-like mechanism that ensures the transient nature of the DNA damage checkpoint.

Roles of increased NUCKS1 expression in endometriosis

BackgroundEndometriosis is still a difficult problem for women. The Nuclear Ubiquitous Casein and cyclin-dependent Kinase Substrate 1 (NUCKS1) gene is located on human chromosome 1q32.1. It encodes the NUCKS1 protein, a 27 kDa nuclear DNA binding protein that plays an important role in cell growth and proliferation. NUCKS1 plays an important role in the development of many diseases. However, its role in endometriosis is unclear.MethodsEctopic endometrial tissues and normal tissue specimens were collected, and the expression of NUCKS1, NF-κB and PI3K was detected by RT-qPCR and immunohistochemistry. Inhibition of NUCKS1 in hEM15A cells, study the changes in cell viability, apoptosis, migration and protein expression by CCK8 assay, flow cytometry, wound-healing assay, western blot and ELISA techniques. The comparison of differences between the two groups was implemented using unpaired sample t test or Mann-whitney U test. One-way analysis of variance or Kruskal-wallis test was used for comparisons among the three groups.Results(1) NUCKS1 is highly expressed in endometriosis tissues. (2) Inhibition of NUCKS1 decreases cell viability and capability of migration, and increases apoptosis in endometriosis cells. (3) Expressions of NF-κB and PI3K are increased in endometriosis tissues, and inhibition of NUCKS1 decreases the expression levels of PI3K and NF-κB in endometriosis cells. (4) Inhibition of NUCKS1 decreases the expression of VEGF.Conclusion(1) NUCKS1 is overexpressed in endometriosis, and inhibition of NUCKS1 inhibits cell viability and capability of migration, and increases apoptosis. (2) NUCKS1 promotes the progress of endometriosis through activating PI3K and NF-κB pathways, and VEFG is also involved in this process.

NUCKS1, a LINC00629-upregulated gene, facilitated osteosarcoma progression and metastasis by elevating asparagine synthesis

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) has been reported to play an oncogenic role in several cancers. However, the biological functions and regulatory mechanism of NUCKS1 in osteosarcoma have not been fully understood. In this study, we reported that NUCKS1 was significantly increased in osteosarcoma. Depletion of NUCKS1 decreased osteosarcoma cell proliferation and metastasis in vivo and in vitro. Overexpression of NUCKS1 accelerated osteosarcoma cell aggressiveness. Mechanistically, NUCKS1 facilitated asparagine (Asn) synthesis by transcriptionally upregulating asparagine synthetase (ASNS) expression and elevating the levels of Asn in osteosarcoma cells, leading to increased cell growth and metastasis. Inhibition of ASNS or reduction of Asn decreased osteosarcoma cell aggressiveness and impaired the promoting effects of NUCKS1 on tumorigenesis and metastasis. Furthermore, we also found that by acting as a sponge for miR-4768-3p, LINC00629 promoted NUCKS1 expression. Collectively, our findings highlight the role of NUCKS1 in regulating asparagine metabolism and reveal that LINC00629 is an important regulator of NUCKS1 that contributes to NUCKS1 upregulation in osteosarcoma.

Micro RNA-365-3p as a Tumor Suppressor in Gastric Cancer: Inhibition of NUCKS1-Mediated PI3K/AKT Signaling and Anti-Tumor Effects

Increasing evidence supports that microRNAs (miRNAs) are essential regulators of tumor initiation and development in a wide range of human malignancies, including gastric cancer (GC). To date, it has been reported that miR-365-3p is abnormally expressed and influences tumor progression in multiple malignancies. However, the roles of miR-365-3p are yet to be known and are worth investigating in GC. Herein, we screened differentially expressed miRNAs in 3 pairs of matched gastric cancerous and adjacent normal specimens. A series of public datasets and collected clinical GC samples were employed to determine the expression profiles of miR-365-3p and its downstream target protein. Furthermore, the effects of miR-365-3p ectopic expression and depletion on GC cell proliferation, invasion and migration were explored in vitro via cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), clone formation and Transwell assays, and in vivo by applying the nude mice models. Dual-luciferase reporter, western blot and a series of rescue assays were carried out to investigate the potential interactions between miR-365-3p and NUCKS1 (Nuclear, casein kinase and cyclin-dependent kinase substrate 1) and downstream signaling pathways of the miR-365-3p/NUCKS1 axis. According to the analyses of the miRNA microarray and bioinformatics, we first identified miR-365-3p dysregulation in GC. Through miRNA qRT-PCR, we revealed that miR-365-3p was downregulated in GC samples and cell lines. Lower level of miR-365-3p was significantly associated with larger tumor size and advanced lymph node metastasis in GC patients. Downregulation of miR-365-3p accelerated, while ectopic miR-365-3p significantly suppressed GC cell proliferation, migration and invasion in vitro and in vivo. Results also showed that overexpression of miR-365-3p in gastric cancer cells significantly reduced the levels of pro-inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), while miR-365-3p knockdown increased their expression. Moreover, mechanistically, knockdown of miR-365-3p promoted tumor cell aggressiveness by enhancing the expression of NUCKS1 and subsequently resulted in the activation of PI3K/AKT signaling pathway in GC. In addition, either NUCKS1 silencing or the inhibition of PI3K/AKT signaling pathway, at least partially, reversed the promotive effects of miR-365-3p knockdown on GC aggressiveness. Altogether, these findings suggest that miR-365-3p inhibits tumor growth and metastasis by attenuating the expression of NUCKS1 and inactivation of its downstream PI3K/AKT signaling, thus, highlighting the potential of miR-365-3p as a therapeutic target in GC.

NUCKS1 promotes the progression of colorectal cancer via activating PI3K/AKT/mTOR signaling pathway.

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) is highly expressed in a variety of malignant tumors and functions as an oncogene; however, its role in colorectal cancer (CRC) remains unclear. We aimed to explore the function and regulatory mechanisms of NUCKS1 and potential therapeutic agents targeting NUCKS1 in CRC. We knocked down and overexpressed NUCKS1 in CRC cells and explored its effects in vitro and in vivo. Flow cytometry, CCK-8, Western blotting, colony formation, immunohistochemistry, in vivo tumorigenic, and transmission electron microscopy analyses were performed to determine the effects of NUCKS1 on CRC cell function. LY294002 was used to examine the mechanism of NUCKS1 expression in CRC cells. Potential therapeutic agents for NUCKS1-high CRC patients were analyzed using the CTRP and PRISM datasets, and the function of selected agents was determined by CCK-8 and Western blotting. We revealed that NUCKS1 was highly expressed in CRC tissues and clinically correlated with poor prognosis in CRC patients. NUCKS1 knockdown induces cell cycle arrest, inhibits CRC cell proliferation, and promotes apoptosis and autophagy. These results were reversed when NUCKS1 was overexpressed. Mechanistically, NUCKS1 exerts a cancer-promoting function by activating the PI3K/AKT/mTOR signaling pathway. This was reversed when LY294002 was used to inhibit the PI3K/AKT pathway. Furthermore, we determined that mitoxantrone exhibited high drug sensitivity in NUCKS1-overexpressing CRC cells. This work demonstrated NUCKS1 plays a crucial role in CRC progression via the PI3K/AKT/mTOR signaling pathway. Additionally, mitoxantrone may be a potential therapeutic agent for CRC treatment. Therefore, NUCKS1 represents a promising anti-tumor therapeutic target.

NUCKS1 promotes breast cancer cell proliferation and metastasis via PI3K/ AKT pathway

Purpose: To investigate the role of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) in breast cancer. 
 Methods: Breast cancer cells were maintained in RPMI-1640 medium containing 10 % fetal bovine serum in a incubator at 37 °C. Cell proliferation was determined by CCK8 and colony formation assays. Flow cytometry and Transwell assays were used to determine cell cycle and metastasis, respectively. 
 Results: Expression of NUCKS1 was significantly elevated in breast cancer (p < 0.01). Overexpression of NUCKS1 significantly increased cell viability (p < 0.01), and promoted proliferation of breast cancer cells. Knockdown of NUCKS1 inhibited cell proliferation, and induced cell cycle arrest at G1 phase. However, overexpression of NUCKS1 promoted cell cycle progression via down-regulation of p21 and up-regulation of cyclin D1 and CDK1. Cell migration and invasion were induced by overexpression of NUCKS1, and suppressed by silencing of NUCKS1. Overexpression of NUCKS1 enhanced p-AKT and p-PI3K expression, while knockdown of NUCKS1 reduced the expression of p-AKT and p-PI3K in breast cancer cells. 
 Conclusion: NUCKS1 promotes breast cancer cell proliferation and metastasis via activation of PI3K/AKT signaling. The silencing of NUCKS1 can be used as a strategy to develop therapies for the management of breast cancer.

NUCKS1 is a highly modified, chromatin-associated protein involved in a diverse set of biological and pathophysiological processes.

The Nuclear Casein and Cyclin-dependent Kinase Substrate 1 (NUCKS1) protein is highly conserved in vertebrates, predominantly localized to the nucleus and one of the most heavily modified proteins in the human proteome. NUCKS1 expression is high in stem cells and the brain, developmentally regulated in mice and associated with several diverse malignancies in humans, including cancer, metabolic syndrome and Parkinson's disease. NUCKS1 function has been linked to modulating chromatin architecture and transcription, DNA repair and cell cycle regulation. In this review, we summarize and discuss the published information on NUCKS1 and highlight the questions that remain to be addressed to better understand the complex biology of this multifaceted protein.

Core control principles of the eukaryotic cell cycle

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin–CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)1,2. However, the principles on which cyclin–CDK complexes organize the temporal order of cell cycle events are contentious3. One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events4–6. A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity7,8. In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events9,10. Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs.

Cyclin J-CDK complexes limit innate immune responses by reducing proinflammatory changes in macrophage metabolism.

Toll-like receptor (TLR) stimulation induces glycolysis and the production of mitochondrial reactive oxygen species (ROS), both of which are critical for inflammatory responses in macrophages. Here, we demonstrated that cyclin J, a TLR-inducible member of the cyclin family, reduced cytokine production in macrophages by coordinately controlling glycolysis and mitochondrial functions. Cyclin J interacted with cyclin-dependent kinases (CDKs), which increased the phosphorylation of a subset of CDK substrates, including the transcription factor FoxK1 and the GTPase Drp1. Cyclin J-dependent phosphorylation of FoxK1 decreased the transcription of glycolytic genes and Hif-1α activation, whereas hyperactivation of Drp1 by cyclin J-dependent phosphorylation promoted mitochondrial fragmentation and impaired the production of mitochondrial ROS. In mice, cyclin J in macrophages limited the growth of tumor xenografts and protected against LPS-induced shock but increased the susceptibility to bacterial infection. Collectively, our findings indicate that cyclin J-CDK signaling promotes antitumor immunity and the resolution of inflammation by opposing the metabolic changes that drive inflammatory responses in macrophages.

MiR-641 Inhibited Cell Proliferation and Induced Apoptosis by Targeting NUCKS1/PI3K/AKT Signaling Pathway in Breast Cancer.

Objective Studies revealed an important role of microRNAs (miRNAs) in multiple cancers, including breast cancer. In the present study, we evaluated the role and function of miR-641 in breast cancer. Methods The expression level of miR-641 in breast cancer cell lines (Hs-578T, MCF7, HCC1937, and MAD-MB-231) was determined by real-time PCR. Functional analyses, including CCK-8 assay, transwell assay, wound-healing assay, and apoptosis detection, were carried out to explore the roles of miRNA-641 in malignant behaviors of breast cancer. Luciferase report assay was used to investigate the regulatory association of miRNA-641 with its potential targets. Results The expression levels of miR-641 were downregulated, while the expression levels of nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) were increased in breast cancer cell lines. The in vitro results showed that miR-641 repressed proliferation and migration/invasion and promoted apoptosis of breast cancer cells. NUCKS1, a positive regulator of phosphatidylinositol-3-kinases (PI3K)/protein-serine-threonine kinase (AKT) pathway, was confirmed as a direct target of miR-641. The of treatment of the PI3K agonist, 740Y-P, could abrogate the antioncogenic potentials of miR-641 in breast cancer cells. Conclusion miR-641 functioned as a tumor suppressor through the PI3K/AKT signaling pathway via targeting NUCKS1 in breast cancer.

Tear miRNA expression analysis reveals miR-203 as a potential regulator of corneal epithelial cells

BackgroundmicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. They are found within cells and in body fluids. Extracellular miRNAs have been shown to associate with the surrounding tissues. Therefore, we predicted that miRNAs in tears may contribute to regulate corneal epithelial cell function. However, information on the miRNA expression profile of tears is limited and the specific functions of tear miRNAs for corneal epithelial cells are still unknown. To study the role of tear miRNAs, we determined which miRNAs are highly expressed in tears and examined the involvement of miRNAs in corneal epithelial cell viability.MethodsmiRNAs extracted from monkey tears and sera were subjected to microarray analysis. miRNAs of which expression levels were higher in tears than in sera were selected, and their expression levels were quantified by quantitative polymerase chain reaction (qPCR). To examine miRNA function, mimics and inhibitors of miRNAs were transfected into human corneal epithelial (HCE-T) cells and incubated for 24 or 48 h. After transfection of miRNA mimics and inhibitors, the viability of HCE-T cells was measured using the water soluble tetrazolium salt (WST) assay, and microarray analysis and qPCR were performed using total RNA extracted from HCE-T cells. siRNAs of the candidate targets for miR-203 were transfected into HCE-T cells and the WST assay was performed. To determine a direct target gene for miR-203, a dual luciferase reporter assay was performed in HCE-T cells using a luciferase reporter plasmid containing 3′-UTR of human IGFBP5.ResultsMicroarray and qPCR analyses showed that miR-184 and miR-203 were expressed significantly more highly in tears than in sera (165,542.8- and 567.8-fold, respectively, p < 0.05). Of these two miRNAs, transfection of a miR-203 mimic significantly reduced the viability of HCE-T cells (p < 0.05), while a miR-203 inhibitor significantly increased this viability (p < 0.05). miR-203 mimic downregulated insulin-like growth factor-binding protein 5 (IGFBP5) and nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), while miR-203 inhibitor upregulated these two genes. Transfection of IGFBP5-siRNA decreased the viability of HCE-T cells. miR-203 mimic significantly diminished the luciferase reporter activity.ConclusionsIn this study, we identified miRNAs that are highly expressed in tears, and the inhibition of miR-203 increases the viability of corneal epithelial cells. Our results suggest that miR-203 contributes to regulating the homeostasis of corneal epithelial cells.

Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

To identify, in myometrial stem/progenitor cells, the presumptive cell of origin for uterine fibroids, substrates of Mediator-associated cyclin dependent kinase 8/19 (CDK8/19), which is known to be disrupted by uterine fibroid driver mutations in Mediator complex subunit 12 (MED12). Experimental study. Academic research laboratory. Women undergoing hysterectomy for uterine fibroids. Stable isotopic labeling of amino acids in cell culture (SILAC) coupled with chemical inhibition of CDK8/19 and downstream quantitative phosphoproteomics and transcriptomic analyses in myometrial stem/progenitor cells. High-confidence Mediator kinase substrates identified by SILAC-based quantitative phosphoproteomics were determined using an empirical Bayes analysis and validated orthogonally by invitro kinase assay featuring reconstituted Mediator kinase modules comprising wild-type or G44D mutant MED12 corresponding to the most frequent uterine fibroid driver mutation in MED12. Mediator kinase-regulated transcripts identified by RNA sequencing were linked to Mediator kinase substrates by computational analyses. A total of 296 unique phosphosites in 166 proteins were significantly decreased (≥ twofold) upon CDK8/19 inhibition, including 118 phosphosites in 71 nuclear proteins representing high-confidence Mediator kinase substrates linked to RNA polymerase II transcription, RNA processing and transport, chromatin modification, cytoskeletal architecture, and DNA replication and repair. Orthogonal validation confirmed a subset of these proteins, including Cut Like Homeobox 1 (CUX1) and Forkhead Box K1 (FOXK1), to be direct targets of MED12-dependent CDK8 phosphorylation in a manner abrogated by the most common uterine fibroid driver mutation (G44D) in MED12, implicating these substrates in disease pathogenesis. Transcriptome-wide profiling of Mediator kinase-inhibited myometrial stem/progenitor cells revealed alterations in cell cycle and myogenic gene expression programs to which Mediator kinase substrates could be linked directly. Among these, CUX1 is an established transcriptional regulator of the cell cycle whose corresponding gene on chromosome 7q is the locus for a recurrent breakpoint in uterine fibroids, linking MED12 and Mediator kinase with CUX1 for the first time in uterine fibroid pathogenesis. FOXK1, a transcriptional regulator of myogenic stem cell fate, was found to be coordinately enriched along with kinase, but not core, Mediator subunits in myometrial stem/progenitor cells compared with differentiated uterine smooth muscle cells. These studies identify a new catalog of pathologically and biologically relevant Mediator kinase substrates implicated in the pathogenesis of MED12 mutation-positive uterine fibroids, and further uncover a biochemical basis to link Mediator kinase activity with CUX1 and FOXK1 in the regulation of myometrial stem/progenitor cell fate.

Docking to a Basic Helix Promotes Specific Phosphorylation by G1-Cdk1.

Cyclins are the activators of cyclin-dependent kinase (CDK) complex, but they also act as docking scaffolds for different short linear motifs (SLiMs) in CDK substrates and inhibitors. According to the unified model of CDK function, the cell cycle is coordinated by CDK both via general CDK activity thresholds and cyclin-specific substrate docking. Recently, it was found that the G1-cyclins of S. cerevisiae have a specific function in promoting polarization and growth of the buds, making the G1 cyclins essential for cell survival. Thus, while a uniform CDK specificity of a single cyclin can be sufficient to drive the cell cycle in some cells, such as in fission yeast, cyclin specificity can be essential in other organisms. However, the known G1-CDK specific LP docking motif, was not responsible for this essential function, indicating that G1-CDKs use yet other unknown docking mechanisms. Here we report a discovery of a G1 cyclin-specific (Cln1,2) lysine-arginine-rich helical docking motif (the K/R motif) in G1-CDK targets involved in the mating pathway (Ste7), transcription (Xbp1), bud morphogenesis (Bud2) and spindle pole body (Spc29, Spc42, Spc110, Sli15) function of S. cerevisiae. We also show that the docking efficiency of K/R motif can be regulated by basophilic kinases such as protein kinase A. Our results further widen the list of cyclin specificity mechanisms and may explain the recently demonstrated unique essential function of G1 cyclins in budding yeast.

NUCKS Promotes the Proliferation, Migration and Invasion of Lung Cancer Cells Through Pi3k/Akt Signalling Pathway.

Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) overexpression has been reported in various types of cancers. The purpose of this study is to clarify the role of NUCKS, underlying the involvement of non-small-cell lung cancer, in the progression of lung cancer. The small interfering ribonucleic acid (siRNA) of NUCKS was transfected into a lung cancer cell line (NCI-H460, A549, NCI-H1299 and NCI-H1975). Functional experiments (MTT assay, Annexin V-FITC/PI double staining assay, colony formation assay, wound healing assay and Transwell assay) were performed to measure the effects of NUCKS on lung cancer cell viability, migration, invasion and apoptosis. NUCKS was found to be up-regulated in lung cancer cells. Knockdown of NUCKS significantly altered lung cancer cell apoptosis, proliferation colony formation, invasion and migration. Moreover, knockdown of NUCKS attenuated the activation of the PI3K/AKT pathway in lung cancer cells. NUCKS was overexpressed in lung cancer cells and played an important role in lung cancer by increasing cell growth through the PI3K/AKT signalling pathway. This in vitro study suggested NUCKS should be evaluated in a clinical setting as a novel biomarker and potential therapeutic target for lung cancer.