Antibody-based assay for N-deacetylase activity of heparan sulfate/heparin N-deacetylase/N-sulfotransferase (NDST): novel characteristics of NDST-1 and -2

A new assay was developed to measure the N-deacetylase activity of the glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs), which are key enzymes in sulfation of heparan sulfate (HS)/heparin. The assay is based on the recognition of NDST-generated N-unsubstituted glucosamine units in Escherichia coli K5 capsular polysaccharide or in HSs by monoclonal antibody JM-403. Substrate specificity and potential product inhibition of the NDST isoforms 1 and 2 were analyzed by comparing lysates of human 293 kidney cells stably transfected with mouse NDST-1 or -2. We found HSs to be excellent substrates for both NDST enzymes. Both NDST-1 and -2 N-deacetylate heparan sulfate from human aorta ( approximately 0.6 sulfate groups/disaccharide) with comparable high efficiency, apparent Km values of 0.35 and 0.76 microM (calculation based on [HexA]) being lower (representing a higher affinity) than those for K5 polysaccharide (13.3 and 4.7 microM, respectively). Comparison of various HS preparations and the unsulfated K5 polysaccharide as substrates indicate that both NDST-1 and -2 can differentially N-sulfate polysaccharides already modified to some extent by various other enzymes involved in HS/heparin synthesis. Both enzymes were equally inhibited by N-sulfated sequences (>or=6 sugar residues) present in N-sulfated K5, N-deacetylated N-resulfated HS, and heparin. Our primary findings were confirmed in the conventional N-deacetylase assay measuring the release of 3H-acetate of radiolabeled K5 or HS as substrates. We furthermore showed that NDST N-deacetylase activity in crude cell/tissue lysates can be partially blocked by endogenous HS/heparin. We speculate that in HS biosynthesis, some NDST variants initiate HS modification/sulfation reactions, whereas other (or the same) NDST isoforms later on fill in or extend already modified HS sequences.     

Role of Cell Surface Glycosaminoglycans of Human T Cells in Human Immunodeficiency Virus Type-1 (HIV-1) Infection

To investigate the role of cell surface glycosaminoglycans (GAGs), including heparan sulfate (HS), on HIV-1 infection in human T cells, HIV-1 binding and infection were determined after treatment of T-cell lines and CD4 + T cells from normal peripheral blood mononuclear cells (PBMC) with GAG-degrading enzyme or a GAG metabolic sulfation inhibitor. Heparitinase I (hep I) and sodium chlorate prevented binding of HIV-1/IIIB to MT-4 cells as revealed by indirect immunofluorescence procedures, thereby inhibiting infection. Hep I was less effective in the binding inhibition of the macrophage-tropic strain HIV-1/SF162 than that of the T-cell line-tropic strain HIV-1/IIIB. The binding of HIV-1/SF162 was about 100-fold less dependent on cell surface HS than HIV-1/IIIB. Human HTLV-I positive T-cell lines expressed more HS than HTLV-I negative T-cell lines or normal CD4 + T cells when stained with anti-HS mAbs against either native or heparitinase-treated HS. With the exception of endo-β-galactosidase (endo-β-gal), GAG-degrading enzymes, including hep I, chondroitinase ABC (chon ABC), chondroitinase AC II (chon AC II) and keratanase, did not prevent the binding of HIV-1/IIIB to CD4+ T cells from normal PBMC. These results indicate that the cell surface HS of human T cells participates in HIV-1 infection by facilitating HIV-1/IIIB binding to MT-4 cells. In particular, the sulfation of HS chains is critical. Since the expression of cell surface HS varies among T cells, which are not consistently sensitive to hep I treatment in HIV-1 binding inhibition, other GAG-like molecules may also be involved.     

Glycosaminoglycans in Hydra magnipapillata (Hydrozoa, Cnidaria): demonstration of chondroitin in the developing nematocyst, the sting organelle, and structural characterization of glycosaminoglycans

The hydrozoan is the simplest organism whose movements are governed by the neuromuscular system, and its de novo morphogenesis can be easily induced by the removal of body parts. These features make the hydrozoan an excellent model for studying the regeneration of tissues in vivo, especially in the nervous system. Although glycosaminoglycans (GAGs) and proteoglycans (PGs) have been implicated in the signaling functions of various growth factors and play critical roles in the development of the central nervous system, the isolation and characterization of GAGs from hydrozoans have never been reported. Here, we characterized GAGs of Hydra magnipapillata. Immunostaining using anti-GAG antibodies showed chondroitin or chondroitin sulfate (CS) in the developing nematocyst, which is a sting organelle specific to cnidarians. The CS-PGs might furnish an environment for assembling nematocyst components, and might themselves be components of nematocysts. Therefore, GAGs were isolated from Hydra and their structural features were examined. A considerable amount of CS, three orders of magnitude less heparan sulfate (HS), but no hyaluronan were found, as in Caenorhabditis elegans. Analysis of the disaccharide composition of HS revealed glucosamine 2-N-sulfation, glucosamine 6-O-sulfation, and uronate 2-O-sulfation. CS contains not only nonsulfated and 4-O-sulfated N-acetylgalactosamine (GalNAc) but also 6-O-sulfated GalNAc. The average molecular size of CS and HS was 110 and 10 kDa, respectively. It has also been established here that CS chains are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide, suggesting that indispensable functions of the linkage region in the synthesis of GAGs have been conserved during evolution.     

BEX2与INI1/hSNF5蛋白的相互作用及其在细胞周期中的功能鉴定

BEX2(Brain expressed X-linked protein 2)分子量约为13 kDa,高表达于人的脑和睾丸中。据报道,该蛋白在胚胎发育中表达量变化巨大,提示该蛋白可能在胚胎发育中具有重要作用,但迄今为止,其功能知之甚少。文章应用酵母双杂交系统,以BEX2为"诱饵"蛋白,筛选发现INI1/hSNF5是BEX2的一个结合蛋白。INI1/hSNF5是SWI/SNF染色质重塑复合体的核心组分,是"表观遗传"分子机制中的重要成员。文章通过体外GST Pull-down实验验证,BEX2与INI1/hSNF5之间的相互作是直接并且特异的。通过进一步的缺失突变分析,表明INI1/hSNF5的两个保守的反向重复序列是BEX2的结合区域。该区域是SNF5与多种蛋白相互作用的结构平台。亚细胞定位分析显示BEX2与INI1/hSNF5都主要集中分布于细胞核,这表明二者之间的相互作用可能参与基因表达调控的过程。文章进一步对此相互作用的功能进行了探讨,发现BEX2通过与INI1/hSNF5的相互作用从而影响细胞周期。     

Defective nitric oxide-dependent, deaminative cleavage of glypican-1 heparan sulfate in Niemann-Pick C1 fibroblasts

Exit of recycling cholesterol from late endosomes is defective in Niemann-Pick C1 (NPC1) and Niemann-Pick C2 (NPC2) diseases. The traffic route of the recycling proteoglycan glypican-1 (Gpc-1) may also involve late endosomes and could thus be affected in these diseases. During recycling through intracellular compartments, the heparan sulfate (HS) side chains of Gpc-1 are deaminatively degraded by nitric oxide (NO) derived from preformed S-nitroso groups in the core protein. We have now investigated whether this NO-dependent Gpc-1 autoprocessing is active in fibroblasts from NPC1 disease. The results showed that Gpc-1 autoprocessing was defective in these cells and, furthermore, greatly depressed in normal fibroblasts treated with U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), a compound widely used to induce cholesterol accumulation. In both cases, autoprocessing was partially restored by treatment with ascorbate which induced NO release, resulting in deaminative cleavage of HS. However, when NO-dependent Gpc-1 autoprocessing is depressed and heparanase-catalyzed degradation of HS remains active, a truncated Gpc-1 with shorter HS chains would prevail, resulting in fewer NO-sensitive sites/proteoglycan. Therefore, addition of ascorbate to cells with depressed autoprocessing resulted in nitration of tyrosines. Nitration was diminished when heparanase was inhibited with suramin or when Gpc-1 expression was silenced by RNAi. Gpc-1 misprocessing in NPC1 cells could thus contribute to neurodegeneration mediated by reactive nitrogen species.     

Overexpression of the 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter 1 increases sulfation of chondroitin sulfate in the apical pathway of MDCK II cells

The canine 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporter1 fused to GFP was stably expressed with a typical Golgi localizationin MDCK II cells (MDCK II-PAPST1). The capacity for PAPS uptakeinto Golgi vesicles was enhanced to almost three times thatof Golgi vesicles isolated from untransfected cells. We havepreviously shown that chondroitin sulfate proteoglycans (CSPGs)are several times more intensely sulfated in the basolateralthan the apical secretory pathway in MDCK II cells (Tveit H,Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes differentmodifications en route to the apical and basolateral surfacesof Madin-Darby canine kidney cells. J Biol Chem. 280:29596–29603).Here we demonstrate that increased availability of PAPS in theGolgi lumen enhances the sulfation of CSPG in the apical pathwayseveral times, while sulfation of CSPGs in the basolateral pathwayshows minor changes. Sulfation of heparan sulfate proteoglycansis essentially unchanged. Our data indicate that CSPG sulfationin the apical pathway of MDCK II cells occurs at suboptimalconditions, either because the sulfotransferases involved havehigh K_m values, or there is a lower PAPS concentration in thelumen of the apical secretory route than in the basolateralcounterpart.     

Concerted mechanism of Swe1/Wee1 regulation by multiple kinases in budding yeast

In eukaryotes, entry into mitosis is induced by cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. In budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is hyperphosphorylated prior to ubiquitin-mediated degradation. Here, we show that Hsl1 and Hsl7 are required for proper localization of Cdc5 (Polo-like kinase homolog) to the bud neck and Cdc5-dependent Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound Cdc28 (Cdk1 homolog) directly phosphorylated Swe1 and this modification served as a priming step to promote subsequent Cdc5-dependent Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 also facilitated Cdc5 localization to the bud neck through the enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 and the polo-box domain of Cdc5. We propose that the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates is an evolutionarily conserved mechanism that is crucial for effectively triggering mitotic entry and other critical mitotic events.     

Ginsenoside Rg1 inhibits tumor necrosis factor-alpha (TNF-alpha)-induced human arterial smooth muscle cells (HASMCs) proliferation

In China, the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The aim of this study was to determine the effects of ginsenoside Rg1 on the proliferation and molecular mechanism in cultured human arterial vascular smooth muscle cell (HASMC) induced by tumor necrosis factor-alpha (TNF-alpha). It was shown that ginsenoside Rg1 significantly inhibited TNF-alpha-induced HASMC proliferation in a dose-dependent manner. Treatment with ginsenoside Rg1, which blocked the cell cycle in the G1-phase, induced a downregulation of cyclin D1 and an upregulation in the expression of p53, p21(WAF/CIP1), and p27(KIP1). MEK inhibitors PD98059, U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not p38-inhibitor SB203580 or JNK-inhibitor SP600125 significantly aggravated ginsenoside Rg1-inhibited HASMC proliferation. Ginsenoside Rg1 markedly inactivated the extracellular signal-regulated kinases (ERK1/2) and protein kinase B (PKB), indicating that the inhibition of ginsenoside Rg1 on HASMC proliferation was associated with ERK and PI3K/PKB pathways. The inactivation of ERK and PI3K/PKB pathways and modulation of cell-cycle proteins by ginsenoside Rg1 may be of importance in inhibition of HASMCs proliferation.     

人源核小体组装蛋白1类似蛋白5（NAP1L5）促进293T细胞增殖

人源NAP1L5为核小体组装蛋白（NAP-1）家族成员,目前功能未知。肝癌研究暗示Nap1l5可能为抑癌基因,抑制细胞增殖;但NAP-1家族其他功能已知的一些成员,可促进细胞增殖和加快周期进程。人源NAP1L5是促进还是抑制细胞增殖,目前未知。本研究里,我们发现过表达Nap1l5促进293T细胞增殖,抑制表达则降低293T细胞增殖速度。细胞周期分析表明：Nap1l5过表达增加G2期、减少G1期细胞比例;抑制Nap1l5表达,则增加G1期、减少G2期细胞比例。我们的研究表明：人源NAP1L5会加快293T细胞周期进程,促进增殖,并且提示原来关于Nap1l5是抑癌基因的推测是不对的。     

Binding to PI(4,5)P2 is indispensable for secretion of B-cell clonogenic HIV-1 matrix protein p17 variants

HIV-1 matrix protein p17 variants (vp17s) derived from non-Hodgkin''s lymphoma (NHL) tissues of HIV-1–seropositive (HIV⁺) patients promote B-cell growth by activating the Akt signaling pathway. It is fundamental to understand the role played by vp17s in producing a microenvironment that fosters lymphoma development and progression. Therefore, we asked whether vp17s could be secreted from infected cells in their biologically active form. In this study, we show that two B-cell growth-promoting vp17s, NHL-a101 and NHL-a102, characterized by amino acid insertions at position 117 to 118 (Ala–Ala) or 125 to 126 (Gly–Asn), respectively, are secreted from HIV-1–infected Jurkat T cells during the active phase of viral replication. Secretion of biologically active vp17s also occurred in HeLa cells nucleofected with a plasmid expressing the entire Gag gene, following proteolytic cleavage of the Gag precursor polyprotein (Pr55^Gag) by cellular aspartyl proteases. Binding of Pr55^Gag to phosphatidylinositol-(4,5)-bisphosphate was indispensable for allowing the unconventional secretion of both wildtype p17 and vp17s. Indeed, here we demonstrate that inhibition of Pr55^Gag binding to phosphatidylinositol-(4,5)-bisphosphate by using neomycin, or its enzymatic depletion achieved by overexpression of 5ptaseIV, significantly impair the secretion of p17s. We also demonstrated that heparan sulfate proteoglycans were involved in tethering p17s at the cell surface. This finding opens up an interesting way for investigating whether tethered p17s on the surface of HIV-1 reservoirs may represent a likely target for immune-mediated killing.     

Structural alteration of cell surface heparan sulfate through the stimulation of the signaling pathway for heparan sulfate 6-O-sulfotransferase-1 in mouse fibroblast cells

Heparan sulfate (HS) is a randomly sulfated polysaccharide that is present on the cell surface and in the extracellular matrix. The sulfated structures of HS were synthesized by multiple HS sulfotransferases, thereby regulating various activities such as growth factor signaling, cell differentiation, and tumor metastasis. Therefore, if the sulfated structures of HS could be artificially controlled, those manipulations would help to understand the various functions depending on HS. However, little knowledge is currently available to realize the mechanisms controlling the expression of such enzymes. In this study, we found that the ratio of 6-O-sulfated disaccharides increased at 3?h after adrenaline stimulation in mouse fibroblast cells. Furthermore, adrenaline-induced up-regulation of HS 6-O-sulfotransferase-1 (6-OST-1) was controlled by Src-ERK1/2 signaling pathway. Finally, inhibiting the signaling pathways for 6-OST-1 intentionally suppressed the adrenaline-induced structural alteration of HS. These observations provide fundamental insights into the understanding of structural alterations in HS by extracellular cues.     

Determination of the warfarin inhibition constant Ki for vitamin K 2,3-epoxide reductase complex subunit-1 (VKORC1) using an in vitro DTT-driven assay

Background

Warfarin directly inhibits vitamin K 2,3-epoxide reductase (VKOR) enzymes. Since the early 1970s, warfarin inhibition of vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), an essential enzyme for proper function of blood coagulation in higher vertebrates, has been studied using an in vitro dithiothreitol (DTT) driven enzymatic assay. However, various studies based on this assay have reported warfarin dose–response data, usually summarized as half-maximal inhibitory concentration (IC₅₀), that vary over orders of magnitude and reflect the broad range of conditions used to obtain VKOR assay data.

Methods

We standardized the implementation of the DTT-driven VKOR activity assay to measure enzymatic Michaelis constants (K_m) and warfarin IC₅₀ for human VKORC1. A data transformation is defined, based on the previously confirmed bi bi ping-pong mechanism for VKORC1, that relates assay condition-dependent IC₅₀ to condition-independent K_i.

Results

Determination of the warfarin K_i specifically depends on measuring both substrate concentrations, both Michaelis constants for the VKORC1 enzyme, and pH in the assay.

Conclusion

The K_i is not equal to the IC₅₀ value directly measured using the DTT-driven VKOR assay.

General significance

In contrast to warfarin IC₅₀ values determined in previous studies, warfarin inhibition expressed as K_i can now be compared between studies, even when the specific DTT-driven VKOR assay conditions differ. This implies that warfarin inhibition reported for wild-type and variant VKORC1 enzymes from previous reports should be reassessed and new determinations of K_i are required to accurately report and compare in vitro warfarin inhibition results.     

Senescence-like changes induced by expression of p21~(Waf1/Cip1) in NIH3T3 cell line

P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/Cip1 involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21Waf1/Cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1 is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/Cip1 expression. Induction of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21Waf1/Cip1-transduced NIH3T3 cells expressed β-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p21Waf1/Cip1 can also induce senescence-like changes in murine cells.     

Premature Cdk1/Cdc5/Mus81 pathway activation induces aberrant replication and deleterious crossover

The error‐free DNA damage tolerance (DDT) pathway is crucial for replication completion and genome integrity. Mechanistically, this process is driven by a switch of templates accompanied by sister chromatid junction (SCJ) formation. Here, we asked if DDT intermediate processing is temporarily regulated, and what impact such regulation may have on genome stability. We find that persistent DDT recombination intermediates are largely resolved before anaphase through a G2/M damage checkpoint‐independent, but Cdk1/Cdc5‐dependent pathway that proceeds via a previously described Mus81‐Mms4‐activating phosphorylation. The Sgs1‐Top3‐ and Mus81‐Mms4‐dependent resolution pathways occupy different temporal windows in relation to replication, with the Mus81‐Mms4 pathway being restricted to late G2/M. Premature activation of the Cdk1/Cdc5/Mus81 pathway, achieved here with phosphomimetic Mms4 variants as well as in S‐phase checkpoint‐deficient genetic backgrounds, induces crossover‐associated chromosome translocations and precocious processing of damage‐bypass SCJ intermediates. Taken together, our results underscore the importance of uncoupling error‐free versus erroneous recombination intermediate processing pathways during replication, and establish a new paradigm for the role of the DNA damage response in regulating genome integrity by controlling crossover timing.     

hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway

hnRNP A1 acts as a critical splicing factor in regulating many alternative splicing events in various physiological and pathophysiological progressions. hnRNP A1 is capable of regulating UVB-induced hdm2 gene alternative splicing according to our previous study. However, the biological function and underlying molecular mechanism of hnRNP A1 in cell survival and cell cycle in response to UVB irradiation are still unclear. In this study, silencing hnRNP A1 expression by siRNA transfection led to decreased cell survival after UVB treatment, while promoting hnRNP A1 by lentiviruse vector resulted in increased cell survival. hnRNP A1 remarkably enhanced PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of Akt, mTOR and P70S6 protein. Inhibition of PI3K/Akt signaling by LY294002 suppressed the expression of hnRNP A1. While mTOR signaling inhibitors, rapamycin and AZD8055, did not influence hnRNP A1 expression in HaCaT cells, suggesting that hnRNP A1 may be an upstream mediator of mTOR signaling. Furthermore, hnRNP A1 could alleviate UVB-provoked cell cycle arrest at G0/G1 phase and promoted cell cycle progression at G2/M phase. Our results indicate that hnRNP A1 promotes cell survival and cell cycle progression following UVB radiation.     

Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor and is refractory to conventional chemotherapy. The aim of this study is therefore to elucidate the mechanism of chemoresistance in ICC which is not fully understood. We generated cisplatin resistant ICC cells via long term exposure to cisplatin and found that these cells are also resistant to 5-fluorouracil (5-FU) and gemcitabine. The chemoresistant cells showed enhanced Bcl-2 expression and reduced Bax expression compared to parental ICC cells. In addition, the resistant cells showed enhanced activation of AKT and extracellular signal-regulated kinase (ERK) 1/2. Inhibition of AKT activation by phosphoinocitide 3-kinase (PI3K) inhibitor LY294002 resulted in reduced Bcl-2 expression and enhanced Bax expression and thus induced apoptosis in the resistant cells, whereas inhibition of ERK1/2 activation by mitogen-activated protein kinase (MEK) inhibitor U0126 did not induce apoptosis without affecting the expression of Bcl-2 and Bax but decreased cell growth. Moreover, the inhibition of AKT or ERK1/2 sensitized the resistant cells to cisplatin and therefore resulted in greatly enhanced cisplatin-induced apoptosis and growth inhibition in the cells. The results indicate that AKT and ERK1/2 signaling mediate chemoresistance in the cells and could be important therapeutic targets for overcoming chemoresistance in ICC.     

Protein kinase B/Akt may regulate G2/M transition in the fertilized mouse egg by changing the localization of p21(Cip1/WAF1)

Protein kinase B (PKB, also called Akt) is known as a serine/threonine protein kinase. Some studies indicate that the Akt signalling pathway strongly promotes G2/M transition in mammalian cell cycle progression, but the mechanism remains to be clarified, especially in the fertilized mouse egg. Here, we report that the expression of Akt at both the protein and mRNA level was highest in G2 phase, accompanied by a peak of Akt activity. In addition, the subcellular localization of p21(Cip1/WAF1) has been proposed to be critical in the cell cycle. Hence, we detected the expression and localization of p21(Cip1/WAF1) after injecting fertilized mouse eggs with Akt mRNA. In one-cell stage fertilized embryos microinjected with mRNA coding for a constitutively active myristoylated Akt (myr-Akt), p21(Cip1/WAF1) was retained in the cytoplasm. Microinjection of mRNA of kinase-deficient Akt(Akt-KD) resulted in nuclear localization of p21(Cip1/WAF1) . Meanwhile, microinjection of different types of Akt mRNA affected the phosphorylation status of p21(Cip1/WAF1) . However, there was no obvious difference in the protein expression of p21(Cip1/WAF1) . Therefore, Akt controls the cell cycle by changing the subcellular localization of p21(Cip1/WAF1) , most likely by affecting the phosphorylation status of p21(Cip1/WAF1) .     

Upregulation of p21(WAF1/Cip1) precedes tumor necrosis factor-induced necrosis-like cell death

The molecular mechanisms mediating death receptor-induced caspase-independent necrotic cell death are still largely unknown. We have previously reported that NIH3T3 cells are sensitized by caspase inhibition to death receptor-induced cytotoxicity leading to a necrosis-like cell death. In addition, we have identified an important role of cell cycle progression for this sensitization effect. Here, we report that tumor necrosis factor-induced necrotic death is preceded by an upregulation of the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). Increased expression of p21(WAF1/Cip1) occurs prior to cell death in the nucleus, where it binds to a cyclin-dependent kinase indicating its functionality. The use of specific pharmacological inhibitors revealed a partial involvement of p38 mitogen-activated protein kinase in the upregulation of p21(WAF1/Cip1). Inhibition of p21(WAF1/Cip1) upregulation prevents a previously observed delay of the cells in the G2/M phase of the cell cycle thereby augmenting, not inhibiting cell death.     

Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations

Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP–Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases.