PP2A function toward mitotic kinases and substrates during the cell cycle

To maintain cellular homeostasis against the demands of the extracellular environment, a precise regulation of kinases and phosphatases is essential. In cell cycle regulation mechanisms, activation of the cyclin-dependent kinase (CDK1) and cyclin B complex (CDK1:cyclin B) causes a remarkable change in protein phosphorylation. Activation of CDK1:cyclin B is regulated by two auto-amplification loops-CDK1:cyclin B activates Cdc25, its own activating phosphatase, and inhibits Wee1, its own inhibiting kinase. Recent biological evidence has revealed that the inhibition of its counteracting phosphatase activity also occurs, and it is parallel to CDK1:cyclin B activation during mitosis. Phosphatase regulation of mitotic kinases and their substrates is essential to ensure that the progression of the cell cycle is ordered. Outlining how the mutual control of kinases and phosphatases governs the localization and timing of cell division will give us a new understanding about cell cycle regulation. [BMB Reports 2013; 46(6): 289-294]     

Human heme oxygenase: cell cycle-dependent expression and DNA microarray identification of multiple gene responses after transduction of endothelial cells

The purpose of the present study was to examine the role of human heme oxygenase (human HO-1) in cell cycle progression following exposure to heme or human HO-1 gene transfer and to identify target genes associated with human HO-1-meditated increases in cell cycle progression using cDNA microarray technology. Heme-induced robust human HO-1 expression in quiescent human microvessel endothelial cells cultured in 1% FBS and the levels of human HO-1 expression progressively declined without a change in the cell cyclin. To identify genes regulated by human HO-1 in the cell cycle, human endothelial cells were transduced with a retroviral vector encoded with human HO-1 gene or an empty vector. Transgene expression and functionality of the recombinant protein were assessed by Western blotting, enzyme activity, carbon monoxide, cGMP production, and cell cycle analysis. Human cDNA gene array and quantitative real-time RT-PCR were used to identify both known and novel differentially expressed genes in cells overexpressing human HO-1. Major findings were upregulation of several genes associated with cell cycle progression, including cyclin E and D; downregulation of cyclin-dependent kinase inhibitors p21 and p27, cyclin-dependent kinases 2, 5, and 6, and monocyte chemoattractant protein-1; and upregulation of growth factors, including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor I (VEGFRI), endothelial growth factor (EGF) and hepatic-derived growth factor (HDGF). These findings identify an array of gene responses to overexpression of human HO-1 and elucidate new aspects of human HO-1 signaling involved in cell growth.     

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor–induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.     

Overexpression of miR-623 suppresses progression of hepatocellular carcinoma via regulating the PI3K/Akt signaling pathway by targeting XRCC5

It has been reported that miR-623 is deregulated in lung adenocarcinoma and inhibits tumor growth and invasion. However, it is unclear whether miR-623 has a role in the progression of hepatocellular carcinoma (HCC). Herein, we found that miR-623 was significantly downregulated in HCC, and that its expression was related to poor clinical outcomes of patients with HCC. Upregulation of miR-623 decreased cell proliferation, viability, migration, and invasion and further promoted apoptosis in 7721, Huh7, and Bel-7402 cells. Moreover, we also observed that miR-623 regulated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt), Wnt/β-catenin, and extracellular regulated protein kinases/c-Jun N-terminal kinase (ERK/JNK) signaling pathways as well as the expression level of related proteins. Further, X-ray repair cross complementing 5 (XRCC5) was a direct target for miR-623, and the suppression of PI3K/Akt, Wnt/β-catenin, and ERK/JNK signaling pathways and cell proliferation and invasion abilities caused by miR-623 in HCC cells was significantly reversed by the upregulation of XRCC5. Collectively, our data suggested that miR-623 suppressed the progression of HCC by regulating the PI3K/Akt, Wnt/β-catenin, and ERK/JNK pathways by targeting XRCC5 in HCC in vitro, indicating that miR-623 may be a target for the therapy of HCC.     

Flavopiridol inhibits lipopolysaccharide-induced TNF-α production through inactivation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway

Flavopiridol is a cyclin-dependent kinase inhibitor and inhibits the growth of various cancer cells. The effect of flavopiridol on lipopolysaccharide (LPS)-induced proinflammatory mediator production was examined in RAW 264.7 macrophage-like cells. Flavopiridol significantly reduced the production of tumor necrosis factor-α and, to a lesser extent, nitric oxide in LPS-stimulated cells. Flavopiridol inhibited the activation of nuclear factor-κB and IκB kinase in response to LPS. Flavopiridol also inhibited the activation of a series of mitogen-activated protein kinases, such as p38, stress-activated protein kinase/c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in response to LPS. However, flavopiridol did not alter the expression of tumor necrosis factor receptor-associated factor 6, myeloid differentiation factor 88 (MyD88) or CD14/toll-like receptor (TLR) 4. Flavopiridol inhibited nitric oxide production induced by a MyD88-dependent TLR2 ligand, but not a MyD88-independent TLR3 ligand. Further, flavopiridol did not alter the phosphorylation of interferon regulatory factor 3 in the MyD88-independent pathway. Therefore, it was suggested that flavopiridol exclusively inhibited the activation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. Flavopiridol might be useful for the prevention of LPS-induced inflammatory response.     

lncRNA HNF1A-AS1 modulates non–small cell lung cancer progression by targeting miR-149-5p/Cdk6

Growing evidence have shown the important regulation of lncRNAs (long noncoding RNAs) in non–small cell lung cancer (NSCLC). lncRNA hepatocyte nuclear factor 1 homeobox A (HNF1A)-antisense RNA 1 (AS1), an “oncogene”, was reported to regulate human tumors progression. However, the molecular mechanism of HNF1A-AS1 involved in the development of NSCLC is still under investigation. In the current study, we found that HNF1A-AS1 was relatively upregulated in both NSCLC patient tissues and cell lines. Functional studies established that overexpression of HNF1A-AS1 promoted cell proliferation, cell cycle, invasion, and migration of NSCLC cells in vitro. The promotion abilities of HNF1A-AS1 on NSCLC cell progression were suppressed via knockdown of HNF1A-AS1. miR-149-5p was then proved to be a novel target of HNF1A-AS1, whose expression was negatively correlated with HNF1A-AS1 in NSCLC patient tissues and cell lines. HNF1A-AS1 increased the expression of cyclin-dependent kinase 6 (Cdk6) via sponging with miR-149-5p. Gain- and loss-of-functional studies indicated that HNF1A-AS1 promoted NSCLC progression partially through inhibition of miR-363-3p and induction of Cdk6. Subcutaneous xenotransplanted tumor model confirmed that interference of HNF1A-AS1 suppressed the tumorigenic ability of NSCLC via upregulation of miR-149-5p and downregulation of Cdk6 in vivo. In conclusion, our findings clarified the biologic significance of the HNF1A-AS1/miR-149-5p/Cdk6 axis in NSCLC progression and provided novel evidence that HNF1A-AS1 may be a new potential therapeutic target for the treatment of NSCLC.     

Acetylation of cyclin-dependent kinase 5 is mediated by GCN5

Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.     

Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster

Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. We have previously shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. In this report, we show that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. Our results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis.     

Effects of the addition of oocyte meiosis-inhibiting drugs on the expression of maturation-promoting factor components and organization of cytoplasmic organelles

The present study evaluated the effects of the blockade of meiosis in bovine oocytes by the cyclin-dependent kinase inhibitors roscovitine (ROS) and butyrolactone-I (BL-I) on nuclear maturation and extracellular signal-regulated kinase 1/2 (ERK1/2), cyclin B1 and p34^cdc2 protein expression and localization. We also evaluated ultrastructural changes in oocytes. Immature oocytes were obtained from slaughtered bovines and divided into: (1) control (oocytes for in vitro maturation only in tissue culture medium-199 for 24 h), (2) oocytes that were treated with 12.5μMROS for 6 h, (3) oocytes that were treated with 50μMBL-I for 6 h and (4) oocytes that were treated with 6.25 μMROS+25 μMBL-I for 6 h. Incubation with inhibitors was followed by the reversal of blockade for 18 h. Oocytes then underwent immunohistochemical analysis to visualize chromatin and assess ERK1/2, cyclin B1 and p34^cdc2 localization/expression, followed by preparation of the cells for ultrastructure analysis by electron microscopy. The groups at 6 h of maturation and before IVM exhibited the lowest number of oocytes in metaphase I. ROS group had the highest number of degenerating oocytes (p < 0.05). After maturation, majority of oocytes were in metaphaseII with no differences among groups (p> 0.05). ERK1/2, cyclin B1 and p34^cdc2 expression differed throughout inhibition and oocyte maturation (p < 0.05). No difference was observed in the localization of these proteins in the ooplasm. No ultrastructural changes in oocytes were observed between treatments, with the exception of treatment with drugs that augmented lipid metabolism (p < 0.05). Results indicate that the effects of CDK1 inhibitors are reversible in bovine oocytes, indicated by nuclear, cytoplasmic, and molecular maturation parameters.