The scaffolding A/Tpd3 subunit and high phosphatase activity are dispensable for Cdc55 function in the Saccharomyces cerevisiae spindle checkpoint and in cytokinesis.
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 tpd3Delta 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.
Koren
Roni
R
Gonda Center of Molecular Microbiology and The Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Bat Galim, Haifa, 31096, Israel.
Rainis
Liat
L
Kleinberger
Tamar
T
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D002384:Catalysis; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D048749:Cytokinesis; D004247:DNA / Q000737:chemistry; D015151:Immunoblotting; D047468:Immunoprecipitation; D008745:Methylation; D016296:Mutagenesis; D009154:Mutation; D015739:Nocodazole / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D008941:Spindle Apparatus; D013997:Time Factors
D000970:Antineoplastic Agents; C071246:CDC55 protein, S cerevisiae; D018797:Cell Cycle Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D015739:Nocodazole