Adenovirus protein E4orf4 induces premature APCCdc20 activation in Saccharomyces cerevisiae by a protein phosphatase 2A-dependent mechanism.

Protein phosphatase 2A (PP2A) has been implicated in cell cycle progression and mitosis; however, the complexity of PP2A regulation via multiple B subunits makes its functional characterization a significant challenge. The human adenovirus protein E4orf4 has been found to induce both high Cdk1 activity and the accumulation of cells in G(2)/M in both mammalian and yeast cells, effects which are largely dependent on the B55/Cdc55 regulatory subunit of PP2A. Thus, E4orf4 represents a unique means by which the function of a specific form of PP2A can be delineated in vivo. In Saccharomyces cerevisiae, only two PP2A regulatory subunits exist, Cdc55 and Rts1. Here, we show that E4orf4-induced toxicity depends on a functional interaction with Cdc55. E4orf4 expression correlates with the inappropriate reduction of Pds1 and Scc1 in S-phase-arrested cells. The unscheduled loss of these proteins suggests the involvement of PP2A(Cdc55) in the regulation of the Cdc20 form of the anaphase-promoting complex (APC). Contrastingly, activity of the Hct1 form of the APC is not induced by E4orf4, as demonstrated by the observed stability of its substrates. We propose that E4orf4, being a Cdc55-specific inhibitor of PP2A, demonstrates the role of PP2A(Cdc55) in regulating APC(Cdc20) activity.

An in vivo assay to quantify stable protein phosphatase 2A (PP2A) heterotrimeric species.

Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. The enzyme is, in fact, largely a collection of varied heterotrimeric species composed of a catalytic (C) subunit and regulatory (B-type) subunit bound together by a structural (A) subunit. One important feature of the C subunit is that its carboxy-terminus can be modified by phosphorylation and methylation. The mechanisms that trigger such posttranslational modifications, as well as their consequences, are still under investigation. However, data collected thus far indicate that these modifications alter the binding to B subunits for an AC dimer, thereby affecting the makeup of the PP2A species in the cell. In this chapter, we describe an in vivo assay for assessing stable PP2A heterotrimer formation that is based on specific subcellular localizations of PP2A heterotrimers. This assay can be used to study the impact of a wide variety of alterations (such as mutations and covalent modifications) on PP2A heterotrimer formation. We specifically describe the use of this assay to quantify the effects of methylation on the stable formation of PP2ARts1p and PP2ACdc55p heterotrimers.

A novel assay for protein phosphatase 2A (PP2A) complexes in vivo reveals differential effects of covalent modifications on different Saccharomyces cerevisiae PP2A heterotrimers.

Protein phosphatase 2A (PP2A) catalytic subunit can be covalently modified at its carboxy terminus by phosphorylation or carboxymethylation. Determining the effects of these covalent modifications on the relative amounts and functions of different PP2A heterotrimers is essential to understanding how these modifications regulate PP2A-controlled cellular processes. In this study we have validated and used a novel in vivo assay for assessing PP2A heterotrimer formation in Saccharomyces cerevisiae: the measurement of heterotrimer-dependent localization of green fluorescent protein-PP2A subunits. This assay relies on the fact that the correct cellular localization of PP2A requires that it be fully assembled. Thus, reduced localization would occur as the result of the inability to assemble a stable heterotrimer. Using this assay, we determined the effects of PP2A C-subunit phosphorylation mimic mutations and reduction or loss of PP2A methylation on the formation and localization of PP2A(B/Cdc55p) and PP2A(B'/Rts1p) heterotrimers. Collectively, our findings demonstrate that phosphorylation and methylation of the PP2A catalytic subunit can influence its function both by regulating the total amount of specific PP2A heterotrimers within a cell and by altering the relative proportions of PP2A(B/Cdc55p) and PP2A(B'/Rts1p) heterotrimers up to 10-fold. Thus, these posttranslational modifications allow flexible, yet highly coordinated, regulation of PP2A-dependent signaling pathways that in turn modulate cell growth and function.

Phosphorylation-dependent regulation of septin dynamics during the cell cycle.

Septins are GTPases involved in cytokinesis. In yeast, they form a ring at the cleavage site. Using FRAP, we show that septins are mobile within the ring at bud emergence and telophase and are immobile during S, G2, and M phases. Immobilization of the septins is dependent on both Cla4, a PAK-like kinase, and Gin4, a septin-dependent kinase that can phosphorylate the septin Shs1/Sep7. Induction of septin ring dynamics in telophase is triggered by the translocation of Rts1, a kinetochore-associated regulatory subunit of PP2A phosphatase, to the bud neck and correlates with Rts1-dependent dephosphorylation of Shs1. In rts1-Delta cells, the actomyosin ring contracts properly but cytokinesis fails. Together our results implicate septins in a late step of cytokinesis and indicate that proper regulation of septin dynamics, possibly through the control of their phosphorylation state, is required for the completion of cytokinesis.

Localization of Saccharomyces cerevisiae protein phosphatase 2A subunits throughout mitotic cell cycle.

Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. This enzyme is a collection of varied heterotrimeric complexes, each composed of a catalytic (C) and regulatory (B) subunit bound together by a structural (A) subunit. To understand the cell cycle dynamics of this enzyme population, we carried out quantitative and qualitative analyses of the PP2A subunits of Saccharomyces cerevisiae. We found the following: the level of each subunit remained constant throughout the cell cycle; there is at least 10 times more of one of the regulatory subunits (Rts1p) than the other (Cdc55p); Tpd3p, the structural subunit, is limiting for both catalytic and regulatory subunit binding. Using green fluorescent protein-tagged forms of each subunit, we monitored the sites of significant accumulation of each protein throughout the cell cycle. The two regulatory subunits displayed distinctly different dynamic localization patterns that overlap with the A and C subunits at the bud tip, kinetochore, bud neck, and nucleus. Using strains null for single subunit genes, we confirmed the hypothesis that regulatory subunits determine sites of PP2A accumulation. Although Rts1p and Tpd3p required heterotrimer formation to achieve normal localization, Cdc55p achieved its normal localization in the absence of either an A or C subunit.

Nitrogen availability alters patterns of accumulation of heat stress-induced proteins in plants.

Mounting evidence suggests that heat-shock proteins (HSPs) play a vital role in enhancing survival at high temperature. There is, however, considerable variation in patterns of HSP production among species, and even among and within individuals of a species. It is not known why this variation exists and to what extent variation in HSPs among organisms might be related to differences in thermotolerance. One possibility is that production of HSPs confers costs and natural selection has worked towards optimizing the cost-to-benefits of HSP synthesis and accumulation. However, the costs of this production have not been determined. If HSP production confers significant nitrogen (N) costs, then we reasoned that plants grown under low-N conditions might accumulate less HSP than high-N plants. Furthermore, if HSPs are related to thermotolerance, then variation in HSPs induced by N (or other factors) might correlate with variation in thermotolerance, here measured as short-term effects of heat stress on net CO2 assimilation and photosystem II (PSII) function. To test these predictions, we grew individuals of a single variety of corn (Zea mays L.) under different N levels and then exposed the plants to acute heat stress. We found that: (1) high-N plants produced greater amounts of mitochondrial Hsp60 and chloroplastic Hsp24 per unit protein than their low-N counterparts; and (2) patterns of HSP production were related to PSII efficiency, as measured by F v/F m. Thus, our results indicate that N availability influences HSP production in higher plants suggesting that HSP production might be resource-limited, and that among other benefits, chloroplast HSPs (e.g., Hsp24) may in some way limit damage to PSII function during heat stress.