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

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Abstract

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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Acknowledgments

The authors thank Dr. Endre Kókai (Department of Medical Chemistry, University of Debrecen, Hungary) and Dr. Barna Peitl (Department of Pharmacology and Pharmacotherapy, University of Debrecen, Hungary) for their help in the generation of OsPP2A B″ polyclonal antibody. This work was supported by a grant from OTKA (The Hungarian Scientific Research Grant), Grant Number: NK-69227 and by The Hungarian Social Renewal Operational Program (TAMOP-4.2.2-A-11/1/KONV-2012-0025 grant). EÁ was supported by the János Bolyai Research Fellowship of the Hungarian Academy of Sciences. GVH thanks Katalin Török and Ildikó Borka for their excellent technical assistance.

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Authors and Affiliations

  1. Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

    Edit Ábrahám, Ping Yu, Dénes Dudits & Gábor V. Horváth

  2. Department of Medical Chemistry, University of Debrecen, Debrecen, Hungary

    Ilona Farkas & Viktor Dombrádi

  3. Laboratory of Proteomics Research, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

    Zsuzsanna Darula & Katalin F. Medzihradszky

  4. Department of Plant Biology and Plant Biochemistry, Faculty of Horticultural Science, Corvinus University of Budapest, Budapest, Hungary

    Erzsébet Varga & Noémi Lukács

  5. Laboratory of Cellular Imaging, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

    Ferhan Ayaydin

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  1. Edit Ábrahám
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Correspondence to Dénes Dudits.

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Edit Ábrahám and Ping Yu have contributed equally to this work.

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Ábrahám, E., Yu, P., Farkas, I. et al. The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1. Plant Mol Biol 87, 125–141 (2015). https://doi.org/10.1007/s11103-014-0265-y

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