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K699 cells were transformed with the CAK1-HA integration vector, pFHE86, containing an 860-base pair fragment of the Cak1 coding region (derived from polymerase chain reaction amplification of genomic DNA) fused to an HA tag (18) followed by an actin terminator, in a vector (pRS305) containing the LEU2 gene [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)]. The COOH-terminal Cak1 sequence was thereby changed from EKP to EKE-MAYPYDVPDYASLGPGL (19).
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85035173052
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note
-
Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; G, Gly; K, Lys; L, Leu; M, Met; P, Pro; S, Ser; V, Val; and Y, Tyr.
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32
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85035172239
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note
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4 and then eluted with buffer A. The eluate was loaded on an 80 ml DEAE-Sepharose column and eluted with a linear NaCI gradient in buffer A. Peak fractions were passed through sulphopropyl (SP) Sepharose, loaded onto Cibacron blue Sepharose (8 ml), and eluted with an NaCI gradient in buffer A. Peak fractions were concentrated by ammonium sulfate precipitation and fractionated on a Pharmacia Superdex 200 gel filtration column (125 ml) equilibrated with buffer A (Fig. 1A). Peak fractions were pooled and loaded onto a heparin-Sepharose column (25 ml) and eluted isocratically with buffer A. The eluate was concentrated with a 1-ml Mono Q column, loaded onto a 1-ml Cibacron blue Sepharose column, and eluted with a linear NaCI gradient (Fig. 1B). Figure 1C illustrates the peak CAK fraction from a heparin-Sepharose column in a separate preparation.
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0029017132
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M. R. Gerber, A. Farrell, R. Deshaies, I. Herskowitz, D. O. Morgan, Proc. Natl. Acad. Sci. U.S.A. 92, 4651 (1995).
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Morgan, D.O.5
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34
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85035173502
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note
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32P]ATP.
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-
35
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0028566645
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18 column. The masses of four tryptic peptides were determined by matrix-assisted laser-desorption time-of-flight mass spectrometry [H. Erdjument-Bromage, M. Lui, D. M. Sabatini, S. H. Snyder, P. Tempst, Protein Sci. 3, 2435 (1994); S. Geromanos, P. Casteels, C. Elicone, M. Powell, P. Tempst, in Techniques in Protein Chemistry, J. W. Crabb, Ed. (Academic Press, San Diego, CA, 1994), vol. 5, pp. 143-150], and two of these peptides were sequenced by automated Edman degradation [P. Tempst, S. Geromanos, C. Elicone, H. Erdjument-Bromage, Methods 6, 248 (1994)]. Small amounts of two peptides from unrelated proteins were also present in the 44-kD fraction and presumably represent contaminants.
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Erdjument-Bromage, H.1
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Tempst, P.5
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36
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85013554753
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J. W. Crabb, Ed. Academic Press, San Diego, CA
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18 column. The masses of four tryptic peptides were determined by matrix-assisted laser-desorption time-of-flight mass spectrometry [H. Erdjument-Bromage, M. Lui, D. M. Sabatini, S. H. Snyder, P. Tempst, Protein Sci. 3, 2435 (1994); S. Geromanos, P. Casteels, C. Elicone, M. Powell, P. Tempst, in Techniques in Protein Chemistry, J. W. Crabb, Ed. (Academic Press, San Diego, CA, 1994), vol. 5, pp. 143-150], and two of these peptides were sequenced by automated Edman degradation [P. Tempst, S. Geromanos, C. Elicone, H. Erdjument-Bromage, Methods 6, 248 (1994)]. Small amounts of two peptides from unrelated proteins were also present in the 44-kD fraction and presumably represent contaminants.
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Geromanos, S.1
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Tempst, P.5
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37
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0002961149
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18 column. The masses of four tryptic peptides were determined by matrix-assisted laser-desorption time-of-flight mass spectrometry [H. Erdjument-Bromage, M. Lui, D. M. Sabatini, S. H. Snyder, P. Tempst, Protein Sci. 3, 2435 (1994); S. Geromanos, P. Casteels, C. Elicone, M. Powell, P. Tempst, in Techniques in Protein Chemistry, J. W. Crabb, Ed. (Academic Press, San Diego, CA, 1994), vol. 5, pp. 143-150], and two of these peptides were sequenced by automated Edman degradation [P. Tempst, S. Geromanos, C. Elicone, H. Erdjument-Bromage, Methods 6, 248 (1994)]. Small amounts of two peptides from unrelated proteins were also present in the 44-kD fraction and presumably represent contaminants.
-
(1994)
Methods
, vol.6
, pp. 248
-
-
Tempst, P.1
Geromanos, S.2
Elicone, C.3
Erdjument-Bromage, H.4
-
38
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85035179099
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note
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32P]ATP.
-
-
-
-
39
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85035176518
-
-
note
-
Purified CDK2-HA, CDK2(T160A)-HA, and CycAΔ171 were prepared as described (3, 18). Cdc28-HA and the mammalian CAK trimer were expressed in Sf9 insect cells with recombinant baculoviruses and purified by conventional chromatography. A GST-Clb2 fusion protein was expressed in bacteria (21) and purified with glutathione affinity resin followed by Hi-Trap Q-Sepharose.
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-
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40
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85035176507
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note
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2 and 1 mM ATP. Activated CDKs were recovered by immunoprecipitation with 12CA5 and tested for histone H1 kinase activity as described (22).
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41
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85035174471
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note
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We thank R. Fisher, H. Chamberlin, and D. Kellogg for reagents; A, Murray for valuable advice; K. Liu and members of the Morgan laboratory for comments on the manuscript; and M. Lui for assistance with protein structural analysis. Supported by grants (to D.O.M.) from the National Institute of General Medical Sciences, the Markey Charitable Trust, and the Rita Allen Foundation, as well as grants from the NSF (to P.T.) and the National Cancer Institute (to the Sloan-Kettering Sequencing Lab), F.H.E. is supported by a University of California President's fellowship. A.F. is supported by a postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada.
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