NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1

Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts

Entry into mitosis depends on the activity of cyclin-dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55δ subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle—high in interphase and suppressed during mitosis. Depletion of PP2A–B55δ (in interphase) from ‘cycling' frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A–B55δ was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A–B55δ in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis

Live-cell imaging RNAi screen identifies PP2A-B55 alpha and importin-beta 1 as key mitotic exit regulators in human cells

Author Posting. © The Authors, 2010. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature Cell Biology 12 (2010): 886-893, doi:10.1038/ncb2092.When vertebrate cells exit mitosis, they reorganize various cellular structures to build functional interphase cells1. This depends on Cdk1 inactivation and subsequent dephosphorylation of its substrates2-4. Members of PP1 and PP2A phosphatase families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5, 6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we used a live imaging assay to screen by RNAi a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identified a trimeric PP2A-B55α complex as a key factor for postmitotic reassembly of the nuclear envelope, the Golgi apparatus, and decondensed chromatin, as well as for mitotic spindle breakdown. Using a chemically-induced mitotic exit assay, we found that PP2A-B55α functions downstream of Cdk1 inactivation. PP2A-B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate histone H1 and it was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor Importin β1, and RNAi depletion of Importin β1 delayed mitotic exit synergistically with PP2A-B55α. This demonstrates that PP2A-B55α and Importin β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells.This work was supported by SNF research grant 3100A0-114120, SNF ProDoc grant PDFMP3_124904, a European Young Investigator (EURYI) award of the European Science Foundation to DWG, and a MBL Summer Research Fellowship by the Evelyn and Melvin Spiegel Fund to DWG, a Roche Ph.D. fellowship to MHAS, and a Mueller fellowship of the Molecular Life Sciences Ph.D. program Zurich to MH. MH and MHAS are fellows of the Zurich Ph.D. Program in Molecular Life Sciences. VJ and JG were supported by grants of the ‘Geconcerteerde OnderzoeksActies’ of the Flemish government, the ‘Interuniversitary Attraction Poles’ of the Belgian Science Policy P6/28 and the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’. AIL is a Wellcome Trust Principal Research Fellow. AAH acknowledges funding by the Max Planck Society, the EU-FP6 integrated project MitoCheck, and the BMBF grant DiGtoP [01GS0859]. Work in the groups of KM and JMP was supported by the EU-FP6 integrated project MitoCheck, Boehringer Ingelheim and by the GEN-AU programme of the Austrian Federal Ministry of Science and Research (Austrian Proteomics Platform III), by MeioSys within the Seventh Framework Programme of the European Commission, and by Chromosome Dynamics, which is funded by the Austrian Science Foundation (FWF)

Modulation of plant HMG-CoA reductase by protein phosphatase 2A: Positive and negative control at a key node of metabolism

The enzyme HMG-CoA reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis, critical not only for normal plant development, but also for the adaptation to demanding environmental conditions. Consistent with this notion, plant HMGR is modulated by many diverse endogenous signals and external stimuli. Protein phosphatase 2A (PP2A) is involved in auxin, abscisic acid, ethylene and brassinosteroid signaling and now emerges as a positive and negative multilevel regulator of plant HMGR, both during normal growth and in response to a variety of stress conditions. The interaction with HMGR is mediated by B" regulatory subunits of PP2A, which are also calcium binding proteins. The new discoveries uncover the potential of PP2A to integrate developmental and calcium-mediated environmental signals in the control of plant HMGR