PP2R1A逆转录病毒的构建及对细胞周期的影响

目的：构建重组PP2R1A基因的逆转录病毒感染HEKTER细胞,观察其定位,验证表达,研究过表达PP2R1A对细胞生长及周期的影响。方法：逆转录病毒载体pMIG-Flag-PP2R1A-IRES-GFP与Pcll0A1瞬时共转染293T细胞,收集病毒感染HEKTER细胞,在荧光显微镜下观察定位,标记荧光单克隆。挑取不同表达强度单克隆做western验证PP2R1A蛋白表达。运用流式细胞分析、体外创伤试验及生长曲线试验研究单克隆细胞的增殖及周期。结果：获得了过表达PP2R1A的单克隆细胞株,PP2R1A在细胞内广泛表达,结合western及细胞试验证实PP2R1A高表达阻滞细胞周期并减慢细胞生长。结论：PP2R1A是丝苏氨酸蛋白磷酸酶PP2A的结构A亚基的a亚型,在细胞内广泛表达。本文成功构建了表达PP2R1A的细胞株,研究发现PP2R1A高表达会影响细胞生长及细胞周期,减缓了细胞增殖。为进一步深入研究PP2R1A对PP2A全酶活性及功能、细胞转化的影响奠定了重要的实验基础。     

PP2R1A 逆转录病毒的构建及对细胞周期的影响

目的:构建重组PP2R1A基因的逆转录病毒感染HEKTER细胞,观察其定位,验证表达,研究过表达PP2R1A对细胞生长及周期的影响。方法:逆转录病毒载体pMIG-Flag-PP2R1A-IRES-GFP与Pcl10A1瞬时共转染293T细胞,收集病毒感染HEKTER细胞,在荧光显微镜下观察定位,标记荧光单克隆。挑取不同表达强度单克隆做western验证PP2R1A蛋白表达。运用流式细胞分析、体外创伤试验及生长曲线试验研究单克隆细胞的增殖及周期。结果:获得了过表达PP2R1A的单克隆细胞株,PP2R1A在细胞内广泛表达,结合western及细胞试验证实PP2R1A高表达阻滞细胞周期并减慢细胞生长。结论:PP2R1A是丝苏氨酸蛋白磷酸酶PP2A的结构A亚基的a亚型,在细胞内广泛表达。本文成功构建了表达PP2R1A的细胞株,研究发现PP2R1A高表达会影响细胞生长及细胞周期,减缓了细胞增殖。为进一步深入研究PP2R1A对PP2A全酶活性及功能、细胞转化的影响奠定了重要的实验基础。     

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]     

Dephosphorylation of Plk1 occurs through PP2A-B55/ENSA/Greatwall pathway during mitotic DNA damage recovery

Recovery from DNA damage is critical for cell survival. However, serious damage cannot be repaired, leading to cell death for prevention of abnormal cell growth. Previously, we demonstrated that 4N-DNA accumulates via the initiation of an abnormal interphase without cytokinesis and that re-replication occurs during a prolonged recovery period in the presence of severe DNA damage in mitotic cells. Mitotic phosphorylated Plk1 is typically degraded during mitotic exit. However, Plk1 has unusually found to be dephosphorylated in mitotic slippage without cytokinesis during recovery from mitotic DNA damage. Here, we investigated how Plk1 dephosphorylation is established during recovery from mitotic DNA damage. Mitotic DNA damage activated ATM and Chk1/2 and repressed Cdk1 and Greatwall protein kinase, followed by PP2A activation through the dissociation of ENSA and PP2A-B55. Interaction between Plk1 and PP2A-B55α or PP2A-B55δ was strongly induced during recovery from mitotic DNA damage. Moreover, the depletion of PP2A-B55α and/or PP2A-B55δ by siRNA transfection led to the recovery of Plk1 phosphorylation and progression of the cell cycle into the G₁ phase. Therefore, to adapt to severe DNA damage, the activated Greatwall/ENSA signaling pathway was repressed by ATM/Chk1/2, even in mitotic cells. Activation of the PP2A-B55 holoenzyme complex induced the dephosphorylation of Plk1 and Cdk1, and finally, mitotic slippage occurred without normal chromosome segregation and cytokinesis.     

FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha

Two different inhibitory domains, N-terminus and central domain, keep FOXM1c almost inactive despite its strong transactivation domain. Here, we demonstrate that cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1 activate FOXM1c. Cyclin E/Cdk2 does not target its transactivation domain or its DNA-binding domain. Instead, its activating effect strictly depends on the presence of either the central domain or the N-terminus of FOXM1c and thus is completely lost if both inhibitory domains are deleted. Cyclin E/Cdk2 activates FOXM1c by releasing its transactivation domain from the repression by these two inhibitory domains. However, it does not directly increase the transactivation potential of the TAD. The DNA-binding is not affected by cyclin E/Cdk2, neither directly nor indirectly. These two activating effects of cyclin E/Cdk2 via central domain and N-terminus are additive. Cyclin A/Cdk2 and cyclin A/Cdk1 show similar characteristics. GSK-3alpha, another proliferation-associated kinase, represses FOXM1c.     

Partial inhibition of Cdk1 in G2 phase overrides the SAC and decouples mitotic events

Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G₂ phase onwards. Addition of low doses of RO3306 in G₂ phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G₂ phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G₂ phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.     

NIRF induces G1 arrest and associates with Cdk2

NIRF is a RING finger protein with a ubiquitin-like domain, a PHD finger, a YDG/SRA domain, and a RING finger domain. Previous study showed that NIRF is a nuclear protein expressed in association with cell proliferation. In this study, we further characterized NIRF functions in cell cycle regulation. Flow cytometric analysis showed that overexpression of NIRF induced an increase in G1 phase cells. Immunoprecipitation and immunoblotting experiments showed that NIRF bound to the inactive Cdk2-cyclin E complex. There existed phosphorylated NIRF in cells, and dephosphorylated NIRF interacted with Cdk2. NIRF was phosphorylated by Cdk2 in vitro. These results suggest that NIRF may participate in the G1/S transition regulation.     

PP2A as a master regulator of the cell cycle

Protein phosphatase 2A (PP2A) plays a critical multi-faceted role in the regulation of the cell cycle. It is known to dephosphorylate over 300 substrates involved in the cell cycle, regulating almost all major pathways and cell cycle checkpoints. PP2A is involved in such diverse processes by the formation of structurally distinct families of holoenzymes, which are regulated spatially and temporally by specific regulators. Here, we review the involvement of PP2A in the regulation of three cell signaling pathways: wnt, mTOR and MAP kinase, as well as the G1→S transition, DNA synthesis and mitotic initiation. These processes are all crucial for proper cell survival and proliferation and are often deregulated in cancer and other diseases.     

Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae

Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G₁, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A^Rts1 either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase.     

Human Cdc25A phosphatase has a non-redundant function in G2 phase by activating Cyclin A-dependent kinases

Cdc25 phosphatases activate Cdk/Cyclin complexes by dephosphorylation and thus promote cell cycle progression. We observed that the peak activity of Cdc25A precedes the one of Cdc25B in prophase and the maximum of Cyclin/Cdk kinase activity. Furthermore, Cdc25A activates both Cdk1-2/Cyclin A and Cdk1/Cyclin B complexes while Cdc25B seems to be involved only in activation of Cdk1/Cyclin B. Concomitantly, repression of Cdc25A led to a decrease in Cyclin A-associated kinase activity and attenuated Cdk1 activation. Our results indicate that Cdc25A acts before Cdc25B - at least in cancer cells, and has non-redundant functions in late G2/early M-phase as a major regulator of Cyclin A/kinase complexes.     

Phosphorylation statuses at different residues of lamin B2, B1, and A/C dynamically and independently change throughout the cell cycle

Lamins, major components of the nuclear lamina, undergo phosphorylation at multiple residues during cell cycle progression, but their detailed phosphorylation kinetics remain largely undetermined. Here, we examined changes in the phosphorylation of major phosphorylation residues (Thr14, Ser17, Ser385, Ser387, and Ser401) of lamin B2 and the homologous residues of lamin B1, A/C during the cell cycle using novel antibodies to the site-specific phosphorylation. The phosphorylation levels of these residues independently changed during the cell cycle. Thr14 and Ser17 were phosphorylated during G₂/M phase to anaphase/telophase. Ser385 was persistently phosphorylated during mitosis to G₁ phase, whereas Ser387 was phosphorylated discontinuously in prophase and G₁ phase. Ser401 phosphorylation was enhanced in the G₁/S boundary. Immunoprecipitation using the phospho-antibodies suggested that metaphase-phosphorylation at Thr14, Ser17, and Ser385 of lamins occurred simultaneously, whereas G₁-phase phosphorylation at Ser385 and Ser387 occurred in distinct pools or with different timings. Additionally, we showed that lamin B2 phosphorylated at Ser17, but not Ser385, Ser387 and Ser401, was exclusively non-ionic detergent soluble, depolymerized forms in growing cells, implicating specific involvement of Ser17 phosphorylation in lamin depolymerization and nuclear envelope breakdown. These results suggest that the phosphorylations at different residues of lamins might play specific roles throughout the cell cycle.     

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.     

The over-expression of p53 H179Y residue mutation causes the increase of cyclin A1 and Cdk4 expression in HELF cells

Down-regulation of p53 expression has been found in a broad range of human cancers and cell proliferation disorders, indicating that p53 plays a key role in cell cycle regulation and tumor suppression. In our current study, we transfected human embryonic lung fibroblast (HELF) cells with pcDNA3-wild-type p53 (pcDNA3-wtp53) plasmid, or pcDNA3-H179Y-mutated p53 (pcDNA3-mtp53) plasmid that mimics the mutation found in some human lung tumors, and further studied the role of p53 in the regulation of cell proliferation. Over expression of wild-type p53 caused cell cycle arrest at G1 phase with reduced cell size, decreased expression of cyclin D3, cyclin E, Cdk2 and Cdk4, and increased expression of p21. In contrast, over expression of H179Y-mutant p53 promoted G1 to S phase transition with enlarged cell size and increased cyclin A1 and Cdk4 expression in HELF cells. These results indicate that mutation at the p53 H179Y residue up-regulates cyclin A1 and Cdk4 expression, and promotes HELF cell proliferation.     

Data mining analysis of the PP2A cell cycle axis in mesothelioma patients

Mesothelioma is an aggressive tumor that affects thousands of people every year. The therapeutic options for patients are limited; hence, a better understanding of mesothelioma biology is crucial to improve patient survival. To find new molecular targets and therapeutic strategies related to the protein phosphatase 2A (PP2A) network, we analyzed the gene expression of known PP2A inhibitors in mesothelioma patient samples. Our analysis disclosed a general overexpression of all PP2A-negative regulators in mesothelioma patients. Moreover, the expression of ANP32E and CIP2A genes, increased in 16% and 11% of cases, positively correlates with the ones of all the other PP2A regulators and the ones of the main cyclins and CDKs, suggesting the existence of a feed-forward loop that might contribute to the mesothelioma progression via PP2A inactivation. Overall, our study indicates the existence of a strategic and targetable axis between PP2A inhibitors (ANP32E and CIP2A) and cell cycle regulators (cyclin B2/CDK1) and provides a valuable rationale for using a personalized combinational therapy approach to improve mesothelioma patient survival.     

Inhibition of Cdk2 Activation by Selected Tyrphostins Causes Cell Cycle Arrest at Late G1 and S Phase

We have previously reported that certain tyrphostins which block EGF-R phosphorylation in cell-free systems fail to do so in intact cells. Nevertheless, we found that this family of tyrphostins inhibits both EGF- and calf serum-induced cell growth and DNA synthesis [Osherov, N.A., Gazit, C., Gilon, and Levitzki, A. (1993). Selective inhibition of the EGF and HER2/Neu receptors by Tyrphostins.J. Biol. Chem.268, 11134–11142.] Now we show that these tyrphostins exert their inhibitory activity even when added at a time when the cells have already passed their restriction point and receptor activation is no longer necessary. AG555 and AG556 arrest 85% of the cells at late G1, whereas AG490 and AG494 cause cells to arrest at late G1 and during S phase. No arrest occurs during G2 or M phase. Further analysis revealed that these tyrphostins act by inhibiting the activation of the enzyme Cdk2 without affecting its levels or its intrinsic kinase activity. Furthermore, they do not alter the association of Cdk2 to cyclin E or cyclin A or to the inhibitory proteins p21 and p27. These compounds also have no effect on the activating phosphorylation of Cdk2 by Cdk2 activating kinase (CAK) and no effect on the catalytic domain of cdc25 phosphatase. These compounds lead to the accumulation of phosphorylated Cdk2 on tyrosine 15 which is most probably the cause for its inhibition leading to cell cycle arrest at G1/S. A structure–activity relationship study defines a very precise pharmacophore, suggesting a unique molecular target not yet identified and which is most probably involved in the regulation of the tyrosine-phosphorylated state of Cdk2. These compounds represent a new class of cell proliferation blockers whose target is Cdk2 activation.     

The greatwall kinase is dominant over PKA in controlling the antagonistic function of ARPP19 in Xenopus oocytes

The small protein ARPP19 plays a dual role during oocyte meiosis resumption. In Xenopus, ARPP19 phosphorylation at S109 by PKA is necessary for maintaining oocytes arrested in prophase of the first meiotic division. Progesterone downregulates PKA, leading to the dephosphorylation of ARPP19 at S109. This initiates a transduction pathway ending with the activation of the universal inducer of M-phase, the kinase Cdk1. This last step depends on ARPP19 phosphorylation at S67 by the kinase Greatwall. Hence, phosphorylated by PKA at S109, ARPP19 restrains Cdk1 activation while when phosphorylated by Greatwall at S67, ARPP19 becomes an inducer of Cdk1 activation. Here, we investigate the functional interplay between S109 and S67-phosphorylations of ARPP19. We show that both PKA and Gwl phosphorylate ARPP19 independently of each other and that Cdk1 is not directly involved in regulating the biological activity of ARPP19. We also show that the phosphorylation of ARPP19 at S67 that activates Cdk1, is dominant over the inhibitory S109 phosphorylation. Therefore our results highlight the importance of timely synchronizing ARPP19 phosphorylations at S109 and S67 to fully activate Cdk1.     

DEPDC1 is a novel cell cycle related gene that regulates mitotic progression

DEPDC1 is a recently identified novel tumor-related gene that is upregulated in several types of cancer and contributes to tumorigenesis. In this study, we have investigated the expression pattern and functional implications of DEPDC1 during cell cycle progression. Expression studies using synchronized cells demonstrated that DEPDC1 is highly expressed in the mitotic phase of the cell cycle. Immunofluorescence assays showed that DEPDC1 is predominantly localized in the nucleus during interphase and is redistributed into the whole cell upon nuclear membrane breakdown in metaphase. Subsequently, siRNA-mediated knockdown of DEPDC1 caused a significant mitotic arrest. Moreover, knockdown of DEPDC1 resulted in remarkable mitotic defects such as abnormal multiple nuclei and multipolar spindle structures accompanied by the upregulation of the A20 gene as well as several cell cycle-related genes such as CCNB1 and CCNB2. Taken together, our current observations strongly suggest that this novel cancerous gene, DEPDC1, plays a pivotal role in the regulation of proper mitotic progression. [BMB Reports 2015; 48(7): 413-418]