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The Serine/Threonine Phosphatase PP5 Interacts with CDC16 and CDC27, Two Tetratricopeptide Repeat-Containing Subunits of the Anaphase-Promoting Complex

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The Serine/Threonine Phosphatase PP5 Interacts with CDC16 and CDC27, Two Tetratricopeptide Repeat-Containing Subunits of the Anaphase-Promoting Complex The serine/threonine phosphatase PP5 interacts with CDC16 and CDC27, two tetratricopeptide repeat-containing subunits of the anaphase-promoting complex. Vincent Ollendorff, Daniel J. Donoghue To cite this version: Vincent Ollendorff, Daniel J. Donoghue. The serine/threonine phosphatase PP5 interacts with CDC16 and CDC27, two tetratricopeptide repeat-containing subunits of the anaphase-promoting complex.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 1997, 272 (51), pp.32011-32018. 10.1074/jbc.272.51.32011. hal-02697780 HAL Id: hal-02697780 https://hal.inrae.fr/hal-02697780 Submitted on 1 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 51, Issue of December 19, pp. 32011–32018, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Serine/Threonine Phosphatase PP5 Interacts with CDC16 and CDC27, Two Tetratricopeptide Repeat-containing Subunits of the Anaphase-promoting Complex* (Received for publication, August 13, 1997, and in revised form, October 6, 1997) Vincent Ollendorff‡ and Daniel J. Donoghue§ From the Department of Chemistry and Biochemistry and the Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0367 The evolutionarily conserved multisubunit complex conserved multisubunit protein complex that recently has been Downloaded from known as the cyclosome or anaphase-promoting com- shown to catalyze the ubiquitination of cyclin B, a necessary plex is involved in catalyzing the ubiquitination of di- step to trigger its degradation by the 26 S proteasome pathway verse substrates in M phase, allowing their destruction during telophase (6, 7). APC is required not only to exit M by the 26 S proteasome and the completion of mitosis. phase but also at an earlier stage to allow the initiation of Three of the eight subunits of the anaphase-promoting anaphase. APC is activated at the metaphase-to-anaphase http://www.jbc.org/ complex (CDC16, CDC23, and CDC27) have been shown transition and apparently catalyzes the ubiquitination of sev- to be phosphorylated in M phase, and their phosphoryl- eral non-cyclin substrates; inhibition of APC leads to met- ation is required for the anaphase-promoting complex aphase arrest (8, 9). In budding yeast it was demonstrated that to be active as a ubiquitin ligase. Several subunits of the PDST1, an inhibitor of chromosome segregation, was a good anaphase-promoting complex contain tetratricopeptide substrate for APC-dependent ubiquitination (10, 11). Destruc- repeats, a protein motif involved in protein/protein in- teractions. PP5 is a serine/threonine phosphatase that tion of this inhibitor is an obligatory step for anaphase to occur at INRA Institut National de la Recherche Agronomique on September 2, 2018 also contains four copies of the tetratricopeptide re- in yeast, and a similar requirement is likely in other organ- peats motif. Here we show by a combination of two- isms. Therefore, by targeting specific substrates for degrada- hybrid analysis and in vitro binding that PP5 interacts tion at different stages of M phase, APC controls the progres- with CDC16 and CDC27, two subunits of the anaphase- sion and completion of mitosis (6). APC is also present at other promoting complex. Only the NH2-terminal domain of stages of the cell cycle and may fulfill other crucial roles, PP5, containing all four tetratricopeptide repeats, is re- particularly in coupling S and M phase, as suggested by genetic quired for this physical interaction. Deletion analysis studies in yeast (12–15). suggests that the site of binding to PP5 is localized to the APC was recently purified from Xenopus laevis and is com- COOH-terminal block of tetratricopeptide repeats in posed of eight subunits (16), two of which (CDC16 and CDC27) CDC16 and CDC27. In addition, indirect immunofluores- have also been characterized in yeast and mammals (9, 17–20). cence showed that PP5 localizes to the mitotic spindle Four of the eight subunits of X. laevis APC contain tetratri- apparatus. The direct interaction of PP5 with CDC16 copeptide repeats (TPR). This degenerate motif of 34 amino and CDC27, as well as its overlapping spindle localiza- acids is widespread in evolution and is often found in tandem tion in mitosis, suggests that PP5 may be involved in the repeats. The TPR motif is thought to form amphipathic a- regulation of the activity of the anaphase-promoting helices that mediate intra- and/or intermolecular protein/pro- complex. tein interactions (17, 21, 22). Indeed, in yeast it has been demonstrated that association between two APC subunits, CDC23 and CDC27, is dependent upon at least part of the TPR The sequential activation of cyclin-dependent kinases con- domain of each protein (17). Immunofluorescence studies in trols progression through the cell cycle (1). Cdc2 and its regu- HeLa cells have shown that a portion of the cellular pool of latory subunit cyclin B have been shown to drive the cell from CDC16 and CDC27 localizes to the spindle/centrosome G into M phase, promoting the multiple events necessary for 2 throughout M phase, where it most likely acts as part of APC to proper chromosome segregation between daughter cells (2). catalyze the ubiquitination of diverse factors (9). Along with kinase activation, proteolysis also plays a major Recent evidence demonstrates that some subunits of Xeno- role during the cell cycle (3–5; for review, see Ref. 6). The pus APC (CDC16, CDC23, and CDC27) become phosphorylated cyclosome, or anaphase-promoting complex (APC),1 is a highly in M phase. Moreover, it was shown that only the phosphoryl- ated form of APC was able to catalyze the ubiquitination of * This work was supported by Grant CA 34456 from the National B-type cyclins and that this activity was abolished by phospha- Institutes of Health. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must tase treatment of M phase-purified APC in a reconstituted therefore be hereby marked “advertisement” in accordance with 18 system (16, 23). However, it remains to be discovered which U.S.C. Section 1734 solely to indicate this fact. kinases and phosphatases control these regulatory phospho- The nucleotide sequence(s) reported in this paper has been submitted rylations and dephosphorylations of APC. to the GenBankTM/EBI Data Bank with accession number(s) AF018262 and AF018263. PP5 is a recently discovered okadaic acid-sensitive, serine/ ‡ Present address: INSERM U1119, 13009 Marseille, France. threonine phosphatase whose substrates and biological func- § To whom correspondence should be addressed: Dept. of Chemistry tions are unknown and which is highly conserved throughout and Biochemistry, University of California, San Diego, 6114 Urey Hall, eukaryotes (24–26). The main structural distinction of PP5 9500 Gilman Dr., La Jolla, California 92093-0367. Tel.: 619-534-2167; compared with other serine/threonine phosphatases lies in the Fax: 619-534-7481; E-mail: [email protected]. 1 The abbreviations used are: APC, anaphase-promoting complex; presence of a TPR domain, containing four repeats, located TPR, tetratricopeptide repeat(s); GST, glutathione S-transferase. NH2-terminal to the catalytic domain (24, 27). Several recent This paper is available on line at http://www.jbc.org 32011 32012 PP5 Interaction with CDC16 and CDC27 reports have shown that this TPR domain of PP5 is in fact between the DNA binding domain LexA and the construct of interest important as an interface for protein/protein interactions. A were cotransformed in the L40 strain with a plasmid encoding a fusion direct interaction has been characterized between this TPR between the activation domain VP16 and a second protein of interest. After growth of the double transformants on selective media (2Trp, domain and the kinase-like domain of the atrial natriuretic 2Leu plates), several individual transformants were tested for their peptide receptor (25). Additionally, a direct in vivo interaction ability to activate the transcription of the two integrated reporter genes between PP5 and the chaperone protein Hsp90 was shown to HIS3 and LACZ. An interaction was scored positive based on the ability involve the TPR domain of PP5 (28, 29). Moreover, these stud- to activate transcription of the HIS3 reporter gene, based on whether ies indicate that PP5 is part of a large heterocomplex involved yeast colonies were able to grow on 2His plates after 3–5 days at 30 °C. in the maturing process of steroid receptors. The presence of The ability to activate the LACZ reporter was also checked by a filter assay as described (30). PP5 in this complex, which also includes Hsp90, the small Expression of PP5-tag from Baculovirus-infected Sf 9 Cells—VSV-G- acidic protein p23, and TPR-containing immunophilins, sug- tagged PP5 (PP5-tag) from pVO120 (BamHI/ClaI) was inserted into gests that PP5 may be intimately involved in protein folding. SLP10 baculovirus vector, and recombinant full-length PP5-tag was In an effort to determine whether PP5 may be involved in expressed in Sf9 cells using baculovirus as described (33). To check for regulating the activity of APC, we have examined its physical expression of PP5-tag, aliquots of PP5-tag Sf9 extracts and SLP10 Sf 9 Downloaded from interaction with two subunits of APC (CDC16 and CDC27) extracts (mock) were analyzed by immunoblotting using the P5D4 an- tibody.
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