Phosphorylation and Activation of 13 S Condensin by Cdc2 in Vitro
10.1126/science.282.5388.487
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

13 S condensin is a multisubunit protein complex essential for mitotic chromosome condensation in Xenopus egg extracts. Purified 13 S condensin introduces positive supercoils into DNA in the presence of topoisomerase I and adenosine triphosphate in vitro. The supercoiling activity of 13 S condensin was regulated by mitosis-specific phosphorylation. Immunodepletion, in vitro phosphorylation, and peptide-mapping experiments indicated that Cdc2 is likely to be the kinase that phosphorylates and activates 13 S condensin. Multiple Cdc2 phosphorylation sites are clustered in the carboxyl-terminal domain of the XCAP-D2 ( Xenopus chromosome-associated polypeptide D2) subunit. These results suggest that phosphorylation of 13 S condensin by Cdc2 may trigger mitotic chromosome condensation in vitro.

Bibliography

Kimura, K., Hirano, M., Kobayashi, R., & Hirano, T. (1998). Phosphorylation and Activation of 13 S Condensin by Cdc2 in Vitro. Science, 282(5388), 487–490.

Authors 4
  1. Keiji Kimura (first)
  2. Michiko Hirano (additional)
  3. Ryuji Kobayashi (additional)
  4. Tatsuya Hirano (additional)
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  13. Immunoaffinity purification of 13 S condensin was done as described (7); for more details see Science Online (www.sciencemag.org). 13 S condensin preparations from two different metaphase extracts [extracts from metaphase II–arrested eggs and extracts activated by cyclin BΔ90 (a nondegradable form of cyclin B)] had identical subunit compositions and supercoiling activities. We do not distinguish between the two preparations and refer to them as the mitotic form throughout this report. Similarly 13 S condensin purified from two different interphase extracts (extracts from eggs activated with the calcium ionophore A23187 and extracts activated in vitro by addition of CaCl 2 ) were indistinguishable. The cell cycle–specific extracts were prepared as described [
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  24. A mixture of 8 S and 13 S condensins was immunoprecipitated with anti–XCAP-E and the XCAP-D2 subunit was gel-purified and processed for microsequencing as described (4). On the basis of two peptide sequences obtained (MEDDFQTPKPPASRK and ENPDIYMAK) (16) we cloned XCAP-D2 cDNA by the reverse transcription–polymerase chain reaction (RT-PCR) two rounds of library screening and nested PCRs. The full-length cDNA was constructed from multiple overlapping cDNAs and sequenced. The cDNA predicted a 1364–amino acid polypeptide with a calculated molecular weight of 154 kD and a pI of 5.46 (GenBank accession number ). A database search identified homologs of unknown functions from yeast (YLR272C) C. elegans () and human ().
  25. Abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
  26. 13 S condensin (∼90 ng) purified from an interphase extract was incubated at 22°C for 30 min in 5 μl of buffer [10 mM Hepes-KCl (pH 7.7) 50 mM KCl 2 mM MgCl 2 0.1 mM CaCl 2 1 mM MgATP 5 mM EGTA 1 mM dithiothreitol and ovalbumin (1 mg/ml)] containing purified Cdc2–cyclin B (∼0.1 ng) (13). A 3-μl aliquot was used in a 5-μl supercoiling reaction (9). Purified Cdc2–cyclin B alone displayed no supercoiling activity. Two other kinases were used as controls. Casein kinase II phosphorylated XCAP-D2 and XCAP-H but none of the three Cdc2 consensus sites of XCAP-D2 were phosphorylated as judged by cross-reactivity to the phosphopeptide antibodies (19). MAP kinase (Erk2) barely phosphorylated the condensin subunits. Neither kinase activated the supercoiling activity of 13 S condensin.
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  33. We thank H. Masuda for anti- Xenopus Cdc2 antiserum M. Solomon for the GST–cyclin B plasmid J. Hemish for her help with the characterization of phosphopeptide antibodies and J. Swedlow and A. Losada for their comments. Supported by NIH grant GM53926 and the Pew Scholars Program in the Biomedical Sciences (T.H.) NIH grant CA45508 (R.K.) and fellowships from the Leukemia Society of America and the Robertson Research Fund (K.K.).
Dates
Type When
Created 23 years ago (July 27, 2002, 5:43 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 12:09 a.m.)
Indexed 2 weeks, 3 days ago (Aug. 7, 2025, 4:39 p.m.)
Issued 26 years, 10 months ago (Oct. 16, 1998)
Published 26 years, 10 months ago (Oct. 16, 1998)
Published Print 26 years, 10 months ago (Oct. 16, 1998)
Funders 0

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@article{Kimura_1998, title={Phosphorylation and Activation of 13 S Condensin by Cdc2 in Vitro}, volume={282}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.282.5388.487}, DOI={10.1126/science.282.5388.487}, number={5388}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Kimura, Keiji and Hirano, Michiko and Kobayashi, Ryuji and Hirano, Tatsuya}, year={1998}, month=oct, pages={487–490} }