Protein phosphatase 2A is required for the initiation of chromosomal DNA replication

Lin, Xiaohong; Walter, Johannes; Scheidtmann, Karl Heinz; Ohst, Kim; Newport, John W.; Walter, Gernot

doi:10.1073/pnas.95.25.14693

Cited by 84 publications

(71 citation statements)

References 46 publications

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“…We also observed inhibition of RPA34 dephosphorylation by okadaic acid (data not shown), which suggests that phosphatase 1A/2A is involved in this dephosphorylation. This is in agreement with the inhibition of DNA replication observed after immunodepletion of phosphatase 2A from Xenopus egg extracts (Lin et al, 1998). We find that hypophosphorylated RPA34 is the only isoform present from the exit of mitosis to the end of S phase; it binds rapidly to chromatin and assembles in discrete nuclear foci, before initiation of DNA synthesis and during the entire process of DNA replication.…”

Section: Discussionsupporting

confidence: 75%

A hypophosphorylated form of RPA34 is a specific component of pre-replication centers

Françon

Lemaı̂tre

Dreyer

et al. 2004

Journal of Cell Science

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IntroductionReplication protein A (RPA) is a stable complex of three different subunits (p70, p34 and p11) that participates in different cellular processes: DNA replication, recombination and repair (Wold, 1997). The RPA70 subunit has a high affinity for single-stranded DNA, but a DNA-binding activity that is associated with the RPA34 and RPA11 subunits (Bochkareva et al., 1998). Cell-cycle-regulated phosphorylation of RPA34 at the G1-S transition has been described (Din et al., 1990;Fang and Newport, 1993;Pan et al., 1994). However, it is not yet clear whether phosphorylation of RPA is involved in the onset of DNA replication (Pan et al., 1995;Philipova et al., 1996), as DNA replication efficiency is not affected by DNAdependent protein kinase (Brush et al., 1994;Lee and Kim, 1995; Pan et al., 1995) or Cdc2 (Henricksen andWold, 1994), two kinases involved in RPA modification.The RPA70 large subunit alone is not sufficient for DNA replication, and the RPA complex cannot be replaced by E. coli single-stranded DNA-binding protein SSB (Dornreiter et al., 1992;Walter and Newport, 2000), suggesting that the RPA34 and RPA11 subunits are necessary for the function of RPA in DNA replication. RPA participates in the synthesis and processing of Okazaki fragments during DNA replication in viruses and in yeast (Mass et al., 1998;Bae et al., 2001). In multicellular organisms, however, its participation in the preinitiation complex is currently unclear. Notably, sites of DNA synthesis are detected as replication foci that co-localize with RPA and the DNA polymerase δ processivity factor PCNA (Adachi and Laemmli, 1992;Dimitrova et al., 1999), but none of the proteins forming the pre-replication complex (e.g. ORC, cdc6, cdt1, MCMs) exhibits such clear localization. The significance of RPA foci is therefore debated.In Xenopus in vitro systems, RPA is present on chromatin before initiation of DNA replication. It first localizes at distinct foci that might be pre-replication centers, but appears to be relocalized evenly throughout the nucleus after initiation of DNA replication (Adachi and Laemmli, 1992). In mammalian cells, RPA localizes to replication foci at the onset of S phase (Brenot-Bosc et al., 1995;Murti et al., 1996), but RPA foci were not observed in early G1 (Dimitrova et al., 1999;Dimitrova and Gilbert, 2000). These observations have led to the proposal that RPA foci in Xenopus may be unrelated to initiation of DNA synthesis and may represent a nonspecific storage of RPA bound to chromatin (Dimitrova et al., 1999).We have investigated the association of the regulatory subunit RPA34 with chromatin during the cell cycle and its participation in pre-replication complexes. We show that the regulatory RPA34 subunit is rapidly dephosphorylated upon mitosis exit, and then associates with chromatin prior to the initiation of DNA replication. RPA34 is detected in its hypophosphorylated form during the whole of S phase and assembles into detergent-resistant foci. Mitosis results in phosphorylation of RPA34 and its loss of chro...

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Section: Discussionsupporting

confidence: 75%

A hypophosphorylated form of RPA34 is a specific component of pre-replication centers

Françon

Lemaı̂tre

Dreyer

et al. 2004

Journal of Cell Science

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“…In addition, PP2A inhibition by okadaic acid can induce apoptosis (51,52). Furthermore, PP2A regulates proliferation by modulating the initiation of DNA replication (53). Therefore, the complete inhibition of PP2A activity can block cell growth and survival.…”

Section: Discussionmentioning

confidence: 99%

The Natural Compound Cantharidin Induces Cancer Cell Death through Inhibition of Heat Shock Protein 70 (HSP70) and Bcl-2-associated Athanogene Domain 3 (BAG3) Expression by Blocking Heat Shock Factor 1 (HSF1) Binding to Promoters

Kim

Kwon

et al. 2013

Journal of Biological Chemistry

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Background: HSF1 is a transcription factor that enhances cancer formation and progression. Results: Cantharidin inhibited the binding of HSF1 to the HSP70 promoter and subsequently blocked HSF1-dependent HSP70 expression. Conclusion:The HSF1-dependent expression of HSP70 and BAG3 is inhibited by cantharidin, causing the down-regulation of antiapoptotic BCL-2 family proteins, especially MCL-1. Significance: This information provides a new target molecule and pathway of cantharidin.

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“…Therefore, here again, it cannot be predicted whether overexpression of PR72 would lead to inhibition or stimulation of dephosphorylation of some PP2A substrates. However, PP2A activity is required for the firing of replication origins (50,51), and this may be mediated by PR72 (21) or another BЉ family member. Binding of PR72 with a relevant substrate in this case likely occurs via the Ca 2ϩ -bound EF1 motif, and Ca 2ϩ binding to EF1 would likely stimulate PP2A activity.…”

Section: Ca 2ϩ -Mediated Regulation Of the Pp2a Bљ/pr72 Subunitmentioning

confidence: 99%

Identification and Functional Analysis of Two Ca2+-binding EF-hand Motifs in the B“/PR72 Subunit of Protein Phosphatase 2A

Janssens

Jordens

Stevens

et al. 2003

Journal of Biological Chemistry

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Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including cell cycle regulation and signal transduction. PP2A is a heterotrimer containing a structural (A) and catalytic (C) subunit, associated with one variable regulatory or targeting B-type subunit, of which three families have been described to date (B/PR55, B/PR61, and B؆/PR72). We identified two functional and highly conserved Ca 2؉ -binding EF-hand motifs in human B؆/PR72 (denoted EF1 and EF2), demonstrating for the first time the ability of Ca 2؉ to interact directly with and regulate PP2A. EF1 and EF2 apparently bind Ca 2؉ with different affinities. Ca 2؉ induces a significant conformational change, which is dependent on the integrity of the motifs. We have further evaluated the effects of Ca 2؉ on subunit composition, subcellular targeting, catalytic activity, and function during the cell cycle of a PR72-containing PP2A trimer (PP2A T72 ) by site-directed mutagenesis of either or both motifs. The results suggest that integrity of EF2 is required for A/PR65 subunit interaction and proper nuclear targeting of PR72, whereas EF1 might mediate the effects of Ca 2؉ on PP2A T72 activity in vitro and is at least partially required for the ability of PR72 to alter cell cycle progression upon forced expression.

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Protein phosphatase 2A is required for the initiation of chromosomal DNA replication

Cited by 84 publications

References 46 publications

A hypophosphorylated form of RPA34 is a specific component of pre-replication centers

A hypophosphorylated form of RPA34 is a specific component of pre-replication centers

The Natural Compound Cantharidin Induces Cancer Cell Death through Inhibition of Heat Shock Protein 70 (HSP70) and Bcl-2-associated Athanogene Domain 3 (BAG3) Expression by Blocking Heat Shock Factor 1 (HSF1) Binding to Promoters

Identification and Functional Analysis of Two Ca2+-binding EF-hand Motifs in the B“/PR72 Subunit of Protein Phosphatase 2A

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