The dual-specificity phosphatase hYVH1 interacts with Hsp70 and prevents heat-shock-induced cell death

hYVH1 [human orthologue of YVH1 (yeast VH1-related phosphatase)] is an atypical dual-specificity phosphatase that is widely conserved throughout evolution. Deletion studies in yeast have suggested a role for this phosphatase in regulating cell growth. However, the role of the human orthologue is unknown. The present study used MS to identify Hsp70 (heat-shock protein 70) as a novel hYVH1-binding partner. The interaction was confirmed using endogenous co-immunoprecipitation experiments and direct binding of purified proteins. Endogenous Hsp70 and hYVH1 proteins were also found to co-localize specifically to the perinuclear region in response to heat stress. Domain deletion studies revealed that the ATPase effector domain of Hsp70 and the zinc-binding domain of hYVH1 are required for the interaction, indicating that this association is not simply a chaperone-substrate complex. Thermal phosphatase assays revealed hYVH1 activity to be unaffected by heat and only marginally affected by non-reducing conditions, in contrast with the archetypical dual-specificity phosphatase VHR (VH1-related protein). In addition, Hsp70 is capable of increasing the phosphatase activity of hYVH1 towards an exogenous substrate under non-reducing conditions. Furthermore, the expression of hYVH1 repressed cell death induced by heat shock, H2O2 and Fas receptor activation but not cisplatin. Co-expression of hYVH1 with Hsp70 further enhanced cell survival. Meanwhile, expression of a catalytically inactive hYVH1 or a hYVH1 variant that is unable to interact with Hsp70 failed to protect cells from the various stress conditions. The results suggest that hYVH1 is a novel cell survival phosphatase that co-operates with Hsp70 to positively affect cell viability in response to cellular insults.     

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.     

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G₂/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G₂/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G₂/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.     

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin‐dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G₂/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV‐encoded E7 oncoprotein and initiates caspase‐dependent apoptosis. Inhibition of the site‐specific phosphorylation of survivin and Bad, occurring at high‐dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G₁/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell‐cycle in G₁ phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF‐7 cells, HeLa cells do not undergo G₁ block after serum starvation, but respond with a slight increase of the ratio of G₁ population. Exposure of G₁‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G₁‐phase, but increases the ratio of S‐ and G₂‐phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G₁‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G₁/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009. © 2009 Wiley‐Liss, Inc.     

PP2ACdc55 regulates G1 cyclin stability

Maintaining accurate progression through the cell cycle requires the proper temporal expression and regulation of cyclins. The mammalian D-type cyclins promote G₁-S transition. D1 cyclin protein stability is regulated through its ubiquitylation and resulting proteolysis catalyzed by the SCF E3 ubiquitin ligase complex containing the F-box protein, Fbx4. SCF E3-ligase-dependent ubiquitylation of D1 is trigged by an increase in the phosphorylation status of the cyclin. As inhibition of ubiquitin-dependent D1 degradation is seen in many human cancers, we set out to uncover how D-type cyclin phosphorylation is regulated. Here we show that in S. cerevisiae, a heterotrimeric protein phosphatase 2A (PP2A^Cdc55) containing the mammalian PPP2R2/PR55 B subunit ortholog Cdc55 regulates the stability of the G₁ cyclin Cln2 by directly regulating its phosphorylation state. Cells lacking Cdc55 contain drastically reduced Cln2 levels caused by degradation due to cdk-dependent hyperphosphorylation, as a Cln2 mutant unable to be phosphorylated by the yeast cdk Cdc28 is highly stable in cdc55-null cells. Moreover, cdc55-null cells become inviable when the SCF^Grr1 activity known to regulate Cln2 levels is eliminated or when Cln2 is overexpressed, indicating a critical relationship between SCF and PP2A functions in regulating cell cycle progression through modulation of G₁-S cyclin degradation/stability. In sum, our results indicate that PP2A is absolutely required to maintain G₁-S cyclin levels through modulating their phosphorylation status, an event necessary to properly transit through the cell cycle.     

Cell cycle checkpoint regulators reach a zillion

Entry into mitosis is regulated by a checkpoint at the boundary between the G₂ and M phases of the cell cycle (G₂/M). In many organisms, this checkpoint surveys DNA damage and cell size and is controlled by both the activation of mitotic cyclin-dependent kinases (Cdks) and the inhibition of an opposing phosphatase, protein phosphatase 2A (PP2A). Misregulation of mitotic entry can often lead to oncogenesis or cell death. Recent research has focused on discovering the signaling pathways that feed into the core checkpoint control mechanisms dependent on Cdk and PP2A. Herein, we review the conserved mechanisms of the G₂/M transition, including recently discovered upstream signaling pathways that link cell growth and DNA replication to cell cycle progression. Critical consideration of the human, frog and yeast models of mitotic entry frame unresolved and emerging questions in this field, providing a prediction of signaling molecules and pathways yet to be discovered.     

Extracellular Signal-regulated Kinase 2 (ERK2) Mediates Phosphorylation and Inactivation of Nuclear Interaction Partner of Anaplastic Lymphoma Kinase (NIPA) at G2/M

NIPA is an F-box-like protein that contributes to the timing of mitotic entry. It targets nuclear cyclin B1 for ubiquitination in interphase, whereas in G₂/M phase, NIPA is inactivated by phosphorylation to allow for cyclin B1 accumulation, a critical event for proper G₂/M transition. We recently specified three serine residues of NIPA and demonstrated a sequential phosphorylation at G₂/M, where initial Ser-354 and Ser-359 phosphorylation is most crucial for SCF^NIPA inactivation. In this study, we identified ERK2 as the kinase responsible for this critical initial phosphorylation step. Using in vitro kinase assays, we found that both ERK1 and ERK2 phosphorylated NIPA with high efficiency. Mutation of either Ser-354 or Ser-359 abolished ERK-dependent NIPA phosphorylation. Pharmacologic inhibition of ERK1/2 in cell lines resulted in decreased NIPA phosphorylation at G₂/M. By combining cell cycle synchronization with stable expression of shRNA targeting either ERK1 or ERK2, we showed that ERK2 but not ERK1 mediated NIPA inactivation at G₂/M. ERK2 knockdown led to a delay at the G₂/M transition, a phenotype also observed in cells expressing a phospho-deficient mutant of NIPA. Thus, our data add to the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation, which may be due in part to the differential ability of these kinases to phosphorylate and inactivate NIPA at G₂/M.     

Varicella-Zoster Virus ORF12 Protein Activates the Phosphatidylinositol 3-Kinase/Akt Pathway To Regulate Cell Cycle Progression

Varicella-zoster virus (VZV) activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and alters cell cycle progression, but the viral protein(s) responsible for these activities is unknown. We previously reported that the VZV open reading frame 12 (ORF12) protein triggers phosphorylation of ERK. Here, we demonstrate that the VZV ORF12 protein also activates the PI3K/Akt pathway to regulate cell cycle progression. Transfection of cells with a plasmid expressing the ORF12 protein induced phosphorylation of Akt, which was dependent on PI3K. Infection of cells with wild-type VZV triggered phosphorylation of Akt, while infection with an ORF12 deletion mutant induced less phosphorylated Akt. The activation of Akt by ORF12 protein was associated with its binding to the p85 subunit of PI3K. Infection of cells with wild-type VZV resulted in increased levels of cyclin B1, cyclin D3, and phosphorylated glycogen synthase kinase 3β (GSK-3β), while infection with the ORF12 deletion mutant induced lower levels of these proteins. Wild-type VZV infection reduced the G₁ phase cell population and increased the M phase cell population, while infection with the ORF12 deletion mutant had a reduced effect on the G₁ and M phase populations. Inhibition of Akt activity with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 reduced the G₁ and M phase differences observed in cells infected with wild-type and ORF12 mutant viruses. In conclusion, we have found that the VZV ORF12 protein activates the PI3K/Akt pathway to regulate cell cycle progression. Since VZV replicates in both dividing (e.g., keratinocytes) and nondividing (neurons) cells, the ability of the VZV ORF12 protein to regulate the cell cycle is likely important for VZV replication in various cell types in the body.     

Matrimony ties Polo down: Can this kinase get free?

Cell cycle progression in female meiotic systems is characterized by the presence of two or more pre-programmed arrests. One such arrest is invariable throughout all species—a lengthy G₂ arrest that separates the end of pachytene (by which time homologous chromosomes have condensed, paired, and undergone recombination1) from nuclear envelope breakdown (NEB). The termination of G₂ arrest (as defined by NEB) and the subsequent entry of the oocyte into prometaphase is regulated by an intracellular signaling cascade whose ultimate feature is the activation of the cyclin B-Cdk1 complex by the Cdc25 phosphatase family. In oocytes, activation of Cdc25 is often mediated by Polo-like kinases (Plks).(2-6) Recent work by Xiang et al. demonstrated that in Drosophila female meiosis, Polo’s role in promoting NEB is regulated by a meiosis-specific stoichiometric inhibitor called Matrimony (Mtrm), which binds to the C-terminal Polo-box domain (PBD) of Polo.(7) In addition to a PBD-binding site, Mtrm contains putative Plk and cyclin B-Cdk1 phosphorylation consensus motifs. These motifs suggest a unique mechanism of Polo inhibition by Mtrm and a possible auto-amplification loop by which cyclin B-Cdk1-mediated destruction or dissociation of Mtrm from Polo allows for rapid and irreversible G₂ exit and entry into prometaphase.     

Phosphorylation of Mixed Lineage Leukemia 5 by Cdc2 Affects Its Cellular Distribution and Is Required for Mitotic Entry

The human mixed lineage leukemia-5 (MLL5) gene is frequently deleted in myeloid malignancies. Emerging evidence suggests that MLL5 has important functions in adult hematopoiesis and the chromatin regulatory network, and it participates in regulating the cell cycle machinery. Here, we demonstrate that MLL5 is tightly regulated through phosphorylation on its central domain at the G₂/M phase of the cell cycle. Upon entry into mitosis, the phosphorylated MLL5 delocalizes from condensed chromosomes, whereas after mitotic exit, MLL5 becomes dephosphorylated and re-associates with the relaxed chromatin. We further identify that the mitotic phosphorylation and subcellular localization of MLL5 are dependent on Cdc2 kinase activity, and Thr-912 is the Cdc2-targeting site. Overexpression of the Cdc2-targeting MLL5 fragment obstructs mitotic entry by competitive inhibition of the phosphorylation of endogenous MLL5. In addition, G₂ phase arrest caused by depletion of endogenous MLL5 can be compensated by exogenously overexpressed full-length MLL5 but not the phosphodomain deletion or MLL5-T912A mutant. Our data provide evidence that MLL5 is a novel cellular target of Cdc2, and the phosphorylation of MLL5 may have an indispensable role in the mitotic progression.     

The Activity of a Wall-Bound Cellulase Is Required for and Is Coupled to Cell Cycle Progression in the Dinoflagellate Crypthecodinium cohnii

Cellulose synthesis, but not its degradation, is generally thought to be required for plant cell growth. In this work, we cloned a dinoflagellate cellulase gene, dCel1, whose activities increased significantly in G₂/M phase, in agreement with the significant drop of cellulose content reported previously. Cellulase inhibitors not only caused a delay in cell cycle progression at both the G₁ and G₂/M phases in the dinoflagellate Crypthecodinium cohnii, but also induced a higher level of dCel1p expression. Immunostaining results revealed that dCel1p was mainly localized at the cell wall. Accordingly, the possible role of cellulase activity in cell cycle progression was tested by treating synchronized cells with exogenous dCelp and purified antibody, in experiments analogous to overexpression and knockdown analyses, respectively. Cell cycle advancement was observed in cells treated with exogenous dCel1p, whereas the addition of purified antibody resulted in a cell cycle delay. Furthermore, delaying the G₂/M phase independently with antimicrotubule inhibitors caused an abrupt and reversible drop in cellulase protein level. Our results provide a conceptual framework for the coordination of cell wall degradation and reconstruction with cell cycle progression in organisms with cell walls. Since cellulase activity has a direct bearing on the cell size, the coupling between cellulase expression and cell cycle progression can also be considered as a feedback mechanism that regulates cell size.     

Human Immunodeficiency Virus Type 1 Vpr Induces Cell Cycle G2 Arrest through Srk1/MK2-Mediated Phosphorylation of Cdc25

Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle G₂ arrest in fission yeast (Schizosaccharomyces pombe) and mammalian cells, suggesting the cellular pathway(s) targeted by Vpr is conserved among eukaryotes. Our previous studies in fission yeast demonstrated that Vpr induces G₂ arrest in part through inhibition of Cdc25, a Cdc2-specific phosphatase that promotes G₂/M transition. The goal of this study was to further elucidate molecular mechanism underlying the inhibitory effect of Vpr on Cdc25. We show here that, similar to the DNA checkpoint controls, expression of vpr promotes subcellular relocalization of Cdc25 from nuclear to cytoplasm and thereby prevents activation of Cdc2 by Cdc25. Vpr-induced nuclear exclusion of Cdc25 appears to depend on the serine/threonine phosphorylation of Cdc25 and the presence of Rad24/14-3-3 protein, since amino acid substitutions of the nine possible phosphorylation sites of Cdc25 with Ala (9A) or deletion of the rad24 gene abolished nuclear exclusion induced by Vpr. Interestingly, Vpr is still able to promote Cdc25 nuclear export in mutants defective in the checkpoints (rad3 and chk1/cds1), the kinases that are normally required for Cdc25 phosphorylation and nuclear exclusion of Cdc25, suggesting that others kinase(s) might modulate phosphorylation of Cdc25 for the Vpr-induced G₂ arrest. We report here that this kinase is Srk1. Deletion of the srk1 gene blocks the nuclear exclusion of Cdc25 caused by Vpr. Overexpression of srk1 induces cell elongation, an indication of cell cycle G₂ delay, in a similar fashion to Vpr; however, no additive effect of cell elongation was observed when srk1 and vpr were coexpressed, indicating Srk1 and Vpr are likely affecting the cell cycle G₂/M transition through the same cellular pathway. Immunoprecipitation further shows that Vpr and Srk1 are part of the same protein complex. Consistent with our findings in fission yeast, depletion of the MK2 gene, a human homologue of Srk1, either by small interfering RNA or an MK2 inhibitor suppresses Vpr-induced cell cycle G₂ arrest in mammalian cells. Collectively, our data suggest that Vpr induces cell cycle G₂ arrest at least in part through a Srk1/MK2-mediated mechanism.     

Ubp-M serine 552 phosphorylation by cyclin-dependent kinase 1 regulates cell cycle progression

In eukaryotic cells, genomic DNA is organized into a chromatin structure, which not only serves as the template for DNA-based nuclear processes, but also as a platform integrating intracellular and extracellular signals. Although much effort has been spent to characterize chromatin modifying/remodeling activities, little is known about cell signaling pathways targeting these chromatin modulators. Here, we report that cyclin-dependent kinase 1 (CDK1) phosphorylates the histone H2A deubiquitinase Ubp-M at serine 552 (S552P), and, importantly, this phosphorylation is required for cell cycle progression. Mass spectrometry analysis confirmed Ubp-M is phosphorylated at serine 552, and in vitro and in vivo assays demonstrated that CDK1/cyclin B kinase is responsible for Ubp-M S552P. Interestingly, Ubp-M S552P is not required for Ubp-M tetramer formation, deubiquitination activity, substrate specificity, or regulation of gene expression. However, Ubp-M S552P is required for cell proliferation and cell cycle G₂/M phase progression. Ubp-M S552P reduces Ubp-M interaction with nuclear export protein CRM1 and facilitates Ubp-M nuclear localization. Therefore, these studies confirm that Ubp-M is phosphorylated at S552 and identify CDK1 as the enzyme responsible for the phosphorylation. Importantly, this study specifically links Ubp-M S552P to cell cycle G₂/M phase progression.     

Okadaic Acid as a Probe to Analyse the Cell Cycle Progression in Plant Cells

Previously we have demonstrated the dynamic change of microtubules (MTs) during cell cycle progression using highly synchronized tobacco BY-2 cells and characterized the specific transition points of MT organization (Hasezawa and Nagata, 1991). In this study the effect of okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, on such changes of MTs during cell cycle was examined. These experiments revealed that cell cycle was arrested before the formation of the preprophase band (PPB), at anaphase and at the border of M/G₁. Although the block at the anaphase seemed to be analogous to that observed in animal cells (Yamashita et al., 1990), the other two blocks were specific to plant cells. It is interesting that these two blocks coincided with the transition points of MT organization, as revealed in the previous study (Hasezawa and Nagata, 1991). Thus it is proposed that phosphorylation is involved in MT organization, although the effect of OA has been shown mainly to be the activation of cdc-2/histone H1 kinase in animal cells. Another inhibitor of protein phosphatase 1 and 2A, calyculin A (CLA), showed very similar effects on the cell cycle progression. The use of such inhibitors to dissect the cell cycle progression of plant cells is discussed.     

Regulation of the Cell Cycle by Protein Phosphatase 2A in Saccharomyces cerevisiae

Protein phosphatase 2A (PP2A) has long been implicated in cell cycle regulation in many different organisms. In the yeast Saccharomyces cerevisiae, PP2A controls cell cycle progression mainly through modulation of cyclin-dependent kinase (CDK) at the G₂/M transition. However, CDK does not appear to be a direct target of PP2A. PP2A affects CDK activity through its roles in checkpoint controls. Inactivation of PP2A downregulates CDK by activating the morphogenesis checkpoint and, consequently, delays mitotic entry. Defects in PP2A also compromise the spindle checkpoint and predispose the cell to an error-prone mitotic exit. In addition, PP2A is involved in controlling the G₁/S transition and cytokinesis. These findings suggest that PP2A functions in many stages of the cell cycle and its effect on cell cycle progression is pleiotropic.     

Multiple cyclin-dependent kinase complexes and phosphatases control G2/M progression in alfalfa cells

Reversible phosphorylation of proteins by kinases and phosphatases plays a key regulatory role in several eukaryotic cellular functions including the control of the division cycle. Increasing numbers of sequence and biochemical data show the involvement of cyclin-dependent kinases (CDKs) and cyclins in regulation of the cell cycle progression in higher plants. The complexity represented by different types of CDKs and cyclins in a single species such as alfalfa, indicates that multicomponent regulatory pathways control G₂/M transition. A set of cdc2-related genes (cdc2Ms A, B, D and F) was expressed in G₂ and M cells. Phosphorylation assays also revealed that at least three kinase complexes (Cdc2Ms A/B, D and F) were successively active in G₂/M cells after synchronization. Interaction between alfalfa mitotic cyclin (Medsa;CycB2;1) and a kinase partner has been reported previously. The present yeast two-hybrid analyses showed differential interaction between defined D-type cyclins and Cdc2Ms kinases functioning in G₂/M phases. Localization of Cdc2Ms F kinase to the preprophase band (PPB), the perinuclear ring in early prophase, the mitotic spindle and the phragmoplast indicated a pivotal role for this kinase in mitotic plant cells. So far limited research efforts have been devoted to the functions of phosphatases in the control of plant cell division. A homologue of dual phosphatase, cdc25, has not been cloned yet from alfalfa; however tyrosine phosphorylation was indicated in the case of Cdc2Ms A kinase and the p^13suc1-bound kinase activity was increased by treatment of this complex with recombinant Drosophila Cdc25. The potential role of serine/threonine phosphatases can be concluded from inhibitor studies based on okadaic acid or endothall. Endothall elevated the kinase activity of p13^suc1-bound fractions in G₂-phase alfalfa cells. These biochemical data are in accordance with observed cytological abnormalities. The present overview with selected original data outlines a conclusion that emphasizes the complexity of G₂/M regulatory events in flowering plants.