Purification and cloning of a protein kinase that phosphorylates and activates the polo-like kinase Plx1

Science

Volumn 282, Issue 5394, 1998, Pages 1701-1704

  Qian, Yue Wei ^a   Erikson, Eleanor ^a   Maller, James L ^a  

^a UNIVERSITY OF COLORADO SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

POLO LIKE KINASE 1; PROTEIN KINASE; PROTEIN KINASE XPLKK1; UNCLASSIFIED DRUG;

AMINO ACID SEQUENCE; ARTICLE; ENZYME ACTIVATION; ENZYME PHOSPHORYLATION; ENZYME PURIFICATION; ENZYME REGULATION; MITOSIS SPINDLE; MOLECULAR CLONING; NONHUMAN; PEPTIDE MAPPING; PREDICTION; PRIORITY JOURNAL; XENOPUS;

EID: 0032573383     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5394.1701     Document Type: Article

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24. 172→Ala mutation), tagged at the COOH-terminus with either Flag or Myc, were lysed, and proteins were immunoprecipitated with antibody to Flag (MZ) or Myc (9E10) (10). The immune complexes were incubated with samples to be assayed in kinase buffer containing 1 mM ATP at 30°C for 30 min. The immune complexes were then washed and assayed for phosphorylation of α-casein (10). Data are expressed as fold activation relative to samples to which control buffer was added.
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28. Two degenerate primers corresponding to the peptide sequences FYDTELETLER and LNEEVAGDPFPSNKPTR (26) were designed: ACIGA(A/G)(C/T)TIGA(A/G)ACI(C/T)TIGA(A/G)(A/C)G and GG(A/G)TCICCIGCIAC(C/T)TC(C/T )TC. PCR was done with 0.5 μg of each primer, 20 ng of Xenopus oocyte cDNA, and 1 U of Taq polymerase (GIBCO) for five cycles at 93°C for 2 min, 54°C for 3 min, and 72°C for 3 min; and 35 cycles at 93°C for 1 min, 54°C for 1 min, and 72°C for 2 min. The reaction generated a 1-kb DNA, which was subcloned, sequenced, and shown to encode the previously determined peptide sequences. A Xenopus oocyte λZAP Express cDNA library was screened with this 1-kb PCR product, and the largest insert was sequenced on both strands. Sequencing was done with an ABI Prism 377 automated DNA fluorescent sequencer (Applied Biosystems) in the DNA Sequencing/Analysis Core Facility at the University of Colorado Cancer Center.
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35. 32P]ATP were treated with progesterone and harvested when about 50% of the oocytes had undergone germinal vesicle breakdown. Plx1 was isolated by immunoprecipitation with antibody to Plx1 (10). Plx1 was phosphorylated in vitro as described (Fig. 3B). Digests were prepared as described [B. G. Gabrielli, M. S. Lee, D. H. Walker, H. Piwnica-Worms, J. L. Maller, J. Biol. Chem. 267, 18040 (1992)] except that chymotrypsin was used and then separated by electrophoresis (pH 8.9, 1000 V, 60 min) in the first dimension and chromatography [pyridine/butanol/acetic acid/water(3.3:5: 1:4)] in the second dimension.
36. Single-letter 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.
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38. 2, 1 mM DTT, 0.01% Brij-35, 10% ethylene glycol), applied it to a Mono S column equilibrated with buffer F containing 20 mM Hepes (pH 7.0), and 2 mM EGTA, and eluted it with a linear gradient up to 1 M NaCl. Fractions containing the xPlkk1 proteins (540 to 600 mM NaCl) were stored in small portions at -80°C. The concentration of protein was determined by SDS gel electrophoresis, Coomassie blue staining, and densitometry, with bovine serum albumin as a standard.
39. Recombinant Plx1 proteins prepared from Sf9 cells (10) were further purified. The eluate from the TALON resin was diluted with four volumes of buffer D and fractionated by hydroxyapatite chromatography (18). Fractions containing the PUI proteins (235 to 265 mM potassium phosphate) were pooled, diluted with four volumes of buffer F, and fractionated by Mono S chromatography. Fractions containing the Plx1 proteins were stored in small portions at -80°C, and the concentration of protein was quantified as described above (28).
40. Portions of the xPlkk1 preparations (60 ng) were treated with 0.15 U of Xenopus PP1 in the presence or absence of 33 μM microcystin at 30°C for 15 min, after which all samples were adjusted to a final concentration of 33 μM microcystin. One unit of PP1 is the amount of enzyme that will release 1.0 nmol of phosphate per minute from phosphorylase a at 30°C. The samples were divided into three portions for immunoblotting xPlkk1 with antibody to Flag (M2) and for assay of phosphorylation and activation of Plx1.
41. 6-tagged xPlkk1 bound to TALON resin was used directly to immunize rabbits, and xPlkk1 antibodies were affinity purified on a column of recombinant xPlkk1 coupled to Affi-gel 10 resin.
42. We thank members of this laboratory for helpful discussions and support, and J. C. Sible for critical reading of the manuscript. The Tissue Culture/Monoclonal Antibody and the DNA Sequencing/Analysis Core Facilities at the University of Colorado Cancer Center were supported by NIH National Cancer Institute Cancer Core support grant (CA46934). Supported in part by a grant from NIH (GM26743). Y.-W.Q. is an Associate and J.L.M. an Investigator of the Howard Hughes Medical Institute.