7 research outputs found
A Chemical Genetic Approach for Covalent Inhibition of Analogue-Sensitive Aurora Kinase
The perturbation of protein kinases with small organic
molecules
is a powerful approach to dissect kinase function in complex biological
systems. Covalent kinase inhibitors that target thiols in the ATP
binding pocket of the kinase domain proved to be ideal reagents for
the investigation of highly dynamic cellular processes. However, due
to the covalent inhibitors' possible off-target reactivities, it is
required that the overall shape of the inhibitor as well as the intrinsic
reactivity of the electrophile are precisely tuned to favor the reaction
with only the desired cysteine. Here we report on the design and biological
characterization of covalent anilinoquinazolines as potent inhibitors
of genetically engineered Aurora kinase in fission yeast
Plk1 Facilitates Cleavage of Human Scc1 by Separase In Vitro
<div><p>(A) Recombinant, <sup>35</sup>S-labeled, wild-type and mutant Scc1 (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030069#pbio-0030069-g001" target="_blank">Figure 1</a>C) tagged with 9xmyc at the C terminus were incubated with human separase. Recombinant human GST-Plk1 was added to the reaction mixtures where indicated. Samples were withdrawn from the reactions at the indicated time points and analyzed by SDS-PAGE followed by immunoblotting (anti-myc) and Phosphorimager analysis (<sup>35</sup>S exposure). Arrows indicate full length Scc1-myc (fl), C- and N-terminal fragments resulting from cleavage at Arg<sup>172</sup> (Ct #1, Nt #1, respectively), and C- and N-terminal fragments resulting from cleavage at Arg<sup>450</sup> (Ct #2, Nt #2, respectively). The lower parts of the membrane or gels were exposed longer than the upper parts. The enhancement of cleavage at Arg<sup>172</sup> by Plk1 can be seen particularly well by comparing the intensities of the N-terminal fragments (Nt #1). Note that in the autoradiographs a band can be seen (particularly clearly in the lanes representing the zero time points) that has almost the same electrophoretic mobility as cleavage product Ct #1. This band is distinct from Ct #1 because it migrates a slightly shorter distance and because it is also present in the absence of separase. This band was therefore not included in the quantification in (B).</p>
<p>(B) Quantification of the abundance of Scc1-myc and the Scc1-myc cleavage fragments in the assay shown in the left autoradiograph of (A). For the quantification, autoradiographs of identical exposure were used. The sum of the intensities of full-length and all cleavage fragments was set to 100%. Signal intensities for N- and C-terminal fragments resulting from cleavage at the same site were summed.</p>
<p>(C) Chromatin fractions were prepared from HeLa cells stably expressing either wild-type Scc1-myc or the mutant Scc1-S<sup>454</sup>A-myc, and were incubated in either interphase or mitotic <i>Xenopus</i> egg extract. Mitosis-specific cleavage of Scc1 was detected by immunoblotting with myc antibodies.</p></div
Phosphorylation of SA2 Is Required for Efficient Resolution of Sister Chromatid Arms during Prometaphase and Metaphase
<div><p>(A) HeLa cells expressing SA2-WT-myc or SA2–12xA-myc were spread on glass slides and chromosomes were stained with Giemsa. Representative cells from SA2 WT-myc or SA2–12xA-myc cell lines after induction with 2 μg/ml doxycycline are shown. Scale bar 10 μm.</p>
<p>(B) HeLa cells were induced to express SA2-WT-myc or SA2–12xA-myc by different amounts of doxycycline as indicated, and processed as in (A). More than 50 cells in prometaphase or metaphase were selected randomly from each sample. The distance between sister chromatids was determined for five chromosomes in each cell and averaged. Light gray bars indicate average values that have been measured in one or two cells, and darker gray bars indicate average values that have been measured in three or more cells. Diamonds indicate the average distance for all cells in a given sample.</p>
<p>(C) Representative immunofluorescence image of normal anaphase in a cell expressing SA2–12xA-myc. The cell was not extracted prior to fixation, so the soluble pool of SA2–12xA-myc is revealed by myc-staining.</p></div
Characterization of HeLa Cell Lines Stably Expressing Wild-Type or Mutant Forms of Human Scc1 and SA2
<div><p>(A) Wild-type Scc1 or SA2, or the indicated mutant proteins (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030069#pbio-0030069-g001" target="_blank">Figure 1</a>C), all tagged with 9xmyc at the C terminus, were stably and inducibly expressed in HeLa tet-on cells. After induction by treatment with 2 μg/ml doxycycline for 1–3 d, cell extracts were prepared from either logarithmically proliferating cells (i, interphase) or from cells arrested in mitosis by nocodazole (m, mitosis), then immunoblotted. In the case of Scc1 cell lines (upper blots), only data from interphase extracts are shown. Exogenous protein was detected by immunoblotting with myc antibodies (lower blots). Since the 9xmyc-tag caused a reduced mobility in SDS-PAGE compared to the endogenous protein, Scc1- and SA2-immunoblots (upper blots) revealed the relative amounts of exogenous and endogenous protein in the different cell lines. The position of molecular weight markers is indicated on the right side.</p>
<p>(B) Extracts were prepared from the different cell lines as indicated. Immunoprecipitation was performed using myc antibodies, followed by SDS-PAGE and silver staining. As a control, the cohesin complex was immunoprecipitated from untransfected HeLa tet-on cells using antibodies to SA2.</p>
<p>(C) Extracts were prepared from SA2-WT-myc or SA2–12xA-myc expressing cells, and fractionated by sucrose density gradient centrifugation (5%–30% sucrose), followed by immunoblotting with antibodies recognizing the proteins indicated on the right (inp. = input/unfractionated sample of the extract).</p></div
The Presence of SA2–12xA on Chromosome Arms Correlates with Cohesion between Sister Chromatid Arms
<div><p>(A) Cells were cultured in the absence or presence of different amounts of doxycycline as indicated. After arrest in nocodazole for 3 h, cells were fixed, spread on glass slides, and stained with Giemsa (photomicrographs, above). Single chromosomes (indicated by a box) are shown at higher magnification in the lower right corners. The number of cells with chromosome arms that had opened (arms open) or that were connected (arms closed) was scored as indicated (bar graphs, below). Scale bar 10 μm.</p>
<p>(B) Whole-cell extracts were prepared from HeLa cells expressing SA2–12xA-myc after treatment with increasing amounts of doxycycline (0, 0.2, and 2.0 μg/ml). The ratio of exogenous SA2–12xA-myc to endogenous SA2 was visualized by immunoblotting with antibodies to SA2. The position of molecular weight markers is indicated on the right.</p></div
Identification of Mitosis-Specific Phosphorylation Sites on Human Cohesin
<div><p>(A and B) Cohesin was immunoprecipitated by antibody 447 (which recognizes SA1 and SA2) from extracts prepared from HeLa cells that were either arrested in S-phase by hydroxyurea (HU) or in mitosis by nocodazole (Noc). Cohesin was eluted by buffer of low pH and analyzed by (A) silver staining and (B) immunoblotting with antibodies to cohesin subunits and phosphorylated threonine (P-Thr).</p>
<p>(C) Schematic representation of the phosphorylation sites on Scc1 and SA2 that were identified by mass spectrometry, and of the mutant versions of the proteins that have been generated. The star indicates a phosphorylation site that was found in both interphase and mitotic Scc1. All SA2 constructs used for in vitro experiments lack the 69 N-terminal amino acids. SA2-WT-myc and SA2–12xA-myc cell lines contain the entire open reading frame of 1,231 amino acids.</p></div
Phosphorylation of SA2 Is Required for Cohesin Dissociation from Chromosome Arms during Prometaphase
<div><p>(A) Logarithmically proliferating HeLa cells expressing SA2-WT-myc or SA2–12xA-myc were extracted prior to fixation, and stained with myc antibodies. In the upper set of images, kinetochores were labeled with human CREST serum, and DNA was counterstained with DAPI. In the lower set of images, only SA2-myc staining is shown.</p>
<p>(B) SA2-myc expression was induced by different amounts of doxycycline (Dox.), and cells were arrested in prometaphase by nocodazole (Noc.) treatment for 3 or 10 h. Cells were spun on glass slides, extracted by detergent, fixed, and processed for immunostaining as in (A). Scale bars 10 μm.</p></div