The Scaffolding A/Tpd3 Subunit and High Phosphatase Activity Are Dispensable for Cdc55 Function in the Saccharomyces cerevisiae Spindle Checkpoint and in Cytokinesis

Abstract

Protein serine/threonine phosphatase 2A (PP2A) is a multifunctional enzyme whose trimeric form consists of a scaffolding A subunit, a catalytic C subunit, and one of several regulatory B subunits (B, B , and B؆). The adenovirus E4orf4 protein associates with PP2A by directly binding the B or B subunits. An interaction with an active PP2A containing the B subunit, or its homologue in yeast, Cdc55, is required for E4orf4-induced apoptosis in mammalian cells and for induction of growth arrest in Saccharomyces cerevisiae. In this work, Cdc55 was randomly mutagenized by low-fidelity PCR amplification, and Cdc55 mutants that lost the ability to transduce the E4orf4 toxic signal in yeast were selected. The mutations obtained by this protocol inhibited the association of Cdc55 with E4orf4, or with the PP2A-AC subunits, or both. Functional analysis revealed that a mutant that does not bind Tpd3, the yeast A subunit, as well as wild type Cdc55 in a tpd3⌬ background, can form a heterodimer with the catalytic subunit. This association requires C subunit carboxyl methylation. The residual phosphatase activity associated with Cdc55 in the absence of Tpd3 is sufficient to maintain a partially active spindle checkpoint and to prevent cytokinesis defects.PP2A 1 is one of the major protein serine/threonine phosphatases in the cell, which plays a role in several cellular processes, including metabolism, transcription, RNA splicing, translation, cell cycle progression, morphogenesis, signal transduction, development, and transformation (1, 2).Several reports indicate that the predominant form of PP2A in cells is a heterotrimer consisting of three subunits. Two of them, the 36-kDa catalytic C subunit and the 63-kDa scaffolding A subunit (PR65), form the core enzyme, and the regulatory B subunit binds the core enzyme to form the holoenzyme. The A and C subunits both exist as two isoforms (␣ and ␤), which are closely related, whereas the B subunit is variable, and its multiple isoforms belong to at least three unrelated gene families, B/B55/PR55 (␣-␦ isoforms), BЈ/B56/PR61 (␣-⑀ isoforms), and BЉ/PR72/PR130/PR59/PR48 (3). The core PP2A enzyme has also been shown to bind other cellular proteins, including striatin and SG2NA (4). Viral proteins, such as the SV40 small t antigen and the polyomavirus small and middle T antigens, can replace the cellular B subunits (5). The various cellular PP2A B subunits target the PP2A holoenzyme to different substrates and dictate its subcellular localization (reviewed in Ref.3).In Saccharomyces cerevisiae, two closely related genes, PPH21 and PPH22, redundantly encode the major PP2A catalytic subunit (6). TPD3 encodes the only A subunit, and two distinct B subunits, encoded by CDC55 and RTS1, are highly homologous to mammalian B and BЈ, respectively (7-9). Mutations of CDC55 are viable, but yield defects in cytokinesis and in the spindle checkpoint and result in abnormal cell morphology. Methylation of the C-terminal leucine residue of the PP2A catalytic subunit by a specific methyltransferase increase...

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…Within the B-type subunit pool, Rts1p (ROX three suppressor 1; the yeast homolog of PR61/B 0 ), is 12Â as abundant as Cdc55p (cell-division-cycle mutant 55; the yeast homolog of PR55/B), indicating that, at any given time, there will always be a free pool of Rts1p and that an as yet undefined mechanism must operate to permit competition between Cdc55p and Rts1p for binding to PP2A D . The same study also confirmed earlier reports demonstrating that, in yeast, subunit stability is not linked to heterotrimer formation [20][21][22][23][24]. However, in Drosophila melanogaster Schneider cells, the absence of all B-type subunits promotes the degradation of the A and C subunits, and vice versa [25,26], which indicates that PP2A enzymes are obligate trimers in this system.…”

Protein Phosphatase 2A B55 and A Regulatory Subunits Interact with Nitrate Reductase and Are Essential for Nitrate Reductase Activation

“…1). Some residual activity due to a complex of the catalytic PP2A subunits with B55, but devoid of A subunits, as has been found for yeast PP2A (Koren et al, 2004) cannot be excluded. Alternatively, a different protein phosphatase, not yet identified, may additionally be involved.…”

Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A)

“…Adult male rats (125-175 g) were anesthetized with pentobarbital (50 mg/kg body weight, administered by intraperitoneal injection) and exsanguinated prior to removal of the liver. For pregnant rats, cesarean sections were performed under pentobarbital anesthesia and fetal (embryonic day 19) livers were harvested before sacrifice. Two-thirds partial hepatectomy was performed on adult male rats (125-175 g) under isofluorane anesthesia as previously described [32].…”

“…Monomeric PP2A catalytic subunit is unstable in Drosophila melanogaster [18] but is stable in yeast [19]. In mammalian cells, there is evidence that PP2A is present in both dimeric and trimeric forms of different compositions [20;21].…”