Plant RBR proteins are phosphorylated in cell cycle-phase dependent manner and the B” regulatory subunit containing OsPP2A holoenzyme mediates the dephosphorylation of OsRBR1

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The thesis work started with the test of specific antibodies used for MsRBR1/OsRBR1 and phospho-MsRBR1/OsRBRs detection. With these antibodies, cell-cycle dependent RBR and phospho- RBR proteins variation in alfalfa and rice cultured cells were checked. Total amount of MsRBR1/OsRBR1 barely changed throughout the cell cycle, while phosphorylated forms of MsRBR1/OsRBR1 proteins showed clear cell cycle dependent changes. In my study, I found that the OsPP2A B” regulatory subunit associates with OsRBR1 but not with OsRBR2. The B pocket of OsRBR1 is essential and sufficient for the interaction between OsPP2A B”. As for B”, none of the truncated version can associate with OsRBR1; the C-terminal of B” is essential but not sufficient for the interacting between these two proteins. Three putative CDK phosphorylation sites of the OsPP2A B” regulatory subunit were verified through LC-MS/MS analysis and the set up of a series of site-directed mutagenesis. The B” regulatory subunit interact with PSTAIRE-motif containing kinases directly and phosphorylate by them, the conclusion comes from the results of co-immunoprecipitation. Elimination of phosphorylation sites in B” did not affect the binding to the PP2A catalytic subunit but did weaken the binding to OsRBR1; the effect was more significant in in vivo yeast two-hybrid system. The phosphorylation elimination of B” regulatory subunit down-regulated the activity of B” containing PP2A heterotrimeric holoenzyme. In contrast, phosphorylation mimicking of B” regulatory subunit up-regulated the activity of B” containing PP2A heterotrimeric holoenzyme. Neither eliminated nor mimicked the phosphorylation of B” regulatory subuint changed the binding strength to PP2A catalytic subunit. The phosphorylation of B” subunit just stimulated the activity of the PP2A complex. It can be postulated that the free Ca2+ ions has the role in regulate the activity of B” subunit since it contains two EF-hand domain. The postulation was verified by the experiments which indicated that the presence of Ca2+ increased the activity of the PP2A holoenzyme and conversely, the absence of Ca2+ (with the chelator, EGTA) inhibited the phosphatase activity of PP2A complex.

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