Phosphorylation and activation of 13S condensin by Cdc2 in vitro

Science

Volumn 282, Issue 5388, 1998, Pages 487-490

  Kimura, Keiji ^a   Hirano, Michiko ^a   Kobayashi, Ryuji ^a   Hirano, Tatsuya ^a  

^a Howard Hughes Medical Institute Cold Spring Harbor Laboratory Cold Spring Harbor   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE TRIPHOSPHATE; DNA; DNA TOPOISOMERASE; POLYPEPTIDE; PROTEIN KINASE; PROTEIN SUBUNIT;

ANIMAL CELL; ARTICLE; CARBOXY TERMINAL SEQUENCE; CHROMOSOME CONDENSATION; CONTROLLED STUDY; DNA SUPERCOILING; EGG; IN VITRO STUDY; MITOSIS; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; XENOPUS;

ADENOSINE TRIPHOSPHATASES; AMINO ACID SEQUENCE; ANIMALS; CDC2 PROTEIN KINASE; CHROMOSOMES; DNA, CIRCULAR; DNA, SUPERHELICAL; DNA-BINDING PROTEINS; ENZYME ACTIVATION; INTERPHASE; MITOSIS; MOLECULAR SEQUENCE DATA; MULTIPROTEIN COMPLEXES; NUCLEIC ACID CONFORMATION; PEPTIDE MAPPING; PHOSPHORYLATION; XENOPUS;

ANIMALIA;

EID: 0032538511     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5388.487     Document Type: Article

References (30)

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13. 2) were indistinguishable. The cell cycle-specific extracts were prepared as described [A. W. Murray, Methods Cell Biol. 36, 581 (1991); T. Hirano and T. J. Mitchison, J. Cell Biol. 115, 1479 (1991)].
14. 2) were indistinguishable. The cell cycle-specific extracts were prepared as described [A. W. Murray, Methods Cell Biol. 36, 581 (1991); T. Hirano and T. J. Mitchison, J. Cell Biol. 115, 1479 (1991)].
15. Supercoiling assay was done as described (7); for more details, see Science Online (www.sciencemag. org). Linking number is a parameter that describes the topological state of a closed circular DNA and corresponds to the number of times that the two chains of DNA twist around one another.
16. K. Kimura and T. Hirano, unpublished data.
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22. A mixture of 8S and 13S 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 pl of 5.46 (GenBank accession number AF067969). A database search identified homologs of unknown functions from yeast (YLR272C), C. elegans (AL021482), and human (D63880).
23. 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.
24. 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 13S condensin.
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26. A synthetic peptide corresponding to the COOH-terminal sequences of XCAP-D2 (CNPTPIRRTARSRAK) was used to prepare an antibody that recognizes both the mitotic and interphase forms of XCAP-D2. To prepare phospho-specific antibodies, we synthesized three phosphopeptides and the corresponding unphosphorylated peptides. The sequences were as follows: DP1, CEDDFQphosphoTPKPPA; DU1, CEDDFQTPKPPA; DP2, CLSEAEphosphoTPKNPT; DU2, CLSEAETPKNPT; DP3, CTPKNPphosphoTPIRRT; DU3, CTPKNPTPIRRT (16). A crude serum raised against the DPx (x = 1, 2, or 3) peptide was passed through an Affi-Gel 10 (Bio-Rad) column conjugated with DUx, and then its flowthrough fraction was loaded onto a second column conjugated with DPx. After extensive washing, phospho-specific antibody was eluted by low pH and used as an affinity-purified anti-DPx. Conjugation of the peptides to a carrier protein and affinity purification of specific antibodies were done as described [K. E. Sawin, T. J. Mitchison, L. G. Wordeman, J. Cell Sci. 102, 303 (1992)].
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30. We thank H. Masuda for anti-Xenopus Cdc2 antiserum, M. Solomon for the GST-cydin 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.).