B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

Jayadeva, Girish; Kurimchak, Alison; Garriga, Judit; Sotillo, Elena; Davis, Anthony J.; Haines, Dale S.; Mumby, Marc C.; Graña, Xavier

doi:10.1074/jbc.m110.162354

Cited by 36 publications

(84 citation statements)

References 28 publications

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“…In mammals, PP2A can modulate the phosphorylation state of the RB-related pocket protein p107 (Rbl1) to promote cell cycle exit in chondrocytes (Jayadeva et al, 2010;Kolupaeva et al, 2008;. Thus, a plausible mechanism for PP2A regulation of the transition to quiescence could be through inhibition of Drosophila retinoblastoma family (Rbf )-mediated repression of E2F/DP transcriptional activity.…”

Section: Inhibition Of Pp2a Leads To Ectopic Cdk2 Activitymentioning

confidence: 99%

Protein phosphatase 2A promotes the transition to G0 during terminal differentiation in Drosophila

Sun

Buttitta

2015

Development

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Protein phosphatase type 2A complex (PP2A) has been known as a tumor suppressor for over two decades, but it remains unclear exactly how it suppresses tumor growth. Here, we provide data indicating a novel role for PP2A in promoting the transition to quiescence upon terminal differentiation in vivo. Using Drosophila eyes and wings as a model, we find that compromising PP2A activity during the final cell cycle prior to a developmentally controlled cell cycle exit leads to extra cell divisions and delays entry into quiescence. By systematically testing the regulatory subunits of Drosophila PP2A, we find that the B56 family member widerborst (wdb) is required for the role of PP2A in promoting the transition to quiescence. Cells in differentiating tissues with compromised PP2A retain high Cdk2 activity when they should be quiescent, and genetic epistasis tests demonstrate that ectopic Cyclin E/Cdk2 activity is responsible for the extra cell cycles caused by PP2A inhibition. The loss of wdb/PP2A function cooperates with aberrantly high Cyclin E protein levels, allowing cells to bypass a robust G0 late in development. This provides an example of how loss of PP2A can cooperate with oncogenic mutations in cancer. We propose that the PP2A complex plays a novel role in differentiating tissues to promote developmentally controlled quiescence through the regulation of Cyclin E/Cdk2 activity.

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Section: Inhibition Of Pp2a Leads To Ectopic Cdk2 Activitymentioning

confidence: 99%

Protein phosphatase 2A promotes the transition to G0 during terminal differentiation in Drosophila

Sun

Buttitta

2015

Development

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Section: Methodsmentioning

confidence: 99%

“…pSG5-puro-Flag-p107 was made by excising the Flag-p107 construct from pCMV-Flag-p107 (20) and inserting it into the pSG5-puro vector. pMSCV-Myc-B55␣ wild type (WT) and pMSCVMyc-B55␣ D197K were described previously (20). pLKO.1 short hairpin RNA (shRNA) Ppp2r2a and scramble plasmids were purchased as a set in bacterial glycerol stock from Sigma (Mission shRNA; SCHLNG-NM_ 028032).…”

Section: Methodsmentioning

confidence: 99%

“…Indeed, we found that PR70 preferentially bound pRB and p130 compared to p107 in in vitro pulldown assays. Altogether, this suggested that, as is the case with CDKs, multiple PP2A holoenzymes may be specialized in mediating dephosphorylation of pocket proteins during the cell cycle or in response to specific cellular cues (20). Indeed, PR70 was found to target pRB for dephosphorylation in response to oxidative stress in a calcium-dependent manner (21).…”

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confidence: 99%

“…For instance, proteomic analysis has identified an excess of 200 distinct PP2A complexes in cells (18). We have recently identified B55␣ as a regulatory B subunit of PP2A that directs p107 and p130 to the PP2A holoenzyme in human cell lines (20). Indeed, B55␣ remains bound to p107 even when excess small t antigen is expressed in cells, which displaces multiple B subunits from the PP2A core dimer.…”

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Activation of p107 by Fibroblast Growth Factor, Which Is Essential for Chondrocyte Cell Cycle Exit, Is Mediated by the Protein Phosphatase 2A/B55α Holoenzyme

Kurimchak

Haines

Garriga

et al. 2013

Molecular and Cellular Biology

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eThe phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible cyclin-dependent kinases (CDKs) and serine/threonine protein phosphatase 2A (PP2A). Two trimeric holoenzymes consisting of the core PP2A catalytic/scaffold dimer and either the B55␣ or PR70 regulatory subunit have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55␣ levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55␣ has not been elucidated. Here we show that fibroblast growth factor 1 (FGF1), which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107-PP2A/B55␣ complexes coinciding with p107 dephosphorylation. Reciprocal solution-based mass spectrometric analysis identified the PP2A/B55␣ complex as a major component in p107 complexes, which also contain E2F/DPs, DREAM subunits, and/or cyclin/CDK complexes. Of note, p107 is one of the preferred partners of B55␣, which also associates with pRB in RCS cells. FGF1-induced dephosphorylation of p107 results in its rapid accumulation in the nucleus and formation of larger complexes containing p107 and enhances its interaction with E2F4 and other p107 partners. Consistent with a key role of B55␣ in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55␣ results in marked dephosphorylation of p107 while B55␣ knockdown results in hyperphosphorylation. More importantly, knockdown of B55␣ dramatically delays FGF1-induced dephosphorylation of p107 and slows down cell cycle exit. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in early recruitment of p107 to the MYC promoter, an FGF1/E2F-regulated gene. Our results suggest a model in which FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylation for several hours after p107 dephosphorylation in maturing chondrocytes.

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The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1

Ábrahám

Farkas

et al. 2014

Plant Mol Biol

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The phosphorylation of plant retinoblastoma-related (RBR) proteins by cyclin-dependent kinases (CDKs) is well documented, but the counteracting phosphatases have not been identified yet. We report here that rice retinoblastoma-related protein-1 (OsRBR1) interacted with the B″ subunit of rice protein phosphatase 2A (OsPP2A B″) and underwent reversible phosphorylation during the cell division cycle. The OsRBR1-OsPP2A B" association required B domain in OsRBR1 and the C-terminal region of OsPP2A B″. We found by immunoprecipitation that OsPP2A B″, OsPP2A catalytic subunit subtype II, PSTAIRE-type CDK and OsRBR1 were in the same protein complex, indicating a physical association between the phosphatase, the kinase and their common substrate. OsPP2A B″ contains three predicted CDK phosphorylation sites: Ser95, Ser102 and Ser119. The in vitro phosphorylation of Ser95 and Ser119 with PSTAIRE-kinases was verified by mass spectrometry. We generated a series of phosphorylation site mutants to mimic the dephosphorylated or phosphorylated states of OsPP2A B″, and confirmed that all of the three predicted sites can be phosphorylated. Yeast two-hybrid experiments suggested that the phosphorylation of OsPP2A B″ promoted the formation of the OsPP2A holoenzyme. A triple phosphorylation mimicking OsPP2A B″ mutant containing holoenzyme showed higher activity in phosphatase assays. Our data collectively show that the phosphatase activity of OsPP2A against OsRBR1 is regulated by the phosphorylation of its B″ regulatory subunit. However, the analysis of the effect of okadaic acid, a phosphatase inhibitor, in rice cell suspension cultures revealed that the dephosphorylation of OsRBR1 was completely inhibited only by high dose (300 nM) of the okadaic acid during the cell cycle progression. Therefore the role of the protein phosphatase 1 should be considered as an additional post translational regulatory component of RBR protein function in higher plants.

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B55α PP2A Holoenzymes Modulate the Phosphorylation Status of the Retinoblastoma-related Protein p107 and Its Activation

Cited by 36 publications

References 28 publications

Protein phosphatase 2A promotes the transition to G0 during terminal differentiation in Drosophila

Protein phosphatase 2A promotes the transition to G0 during terminal differentiation in Drosophila

Activation of p107 by Fibroblast Growth Factor, Which Is Essential for Chondrocyte Cell Cycle Exit, Is Mediated by the Protein Phosphatase 2A/B55α Holoenzyme

The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1

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