Volume 2, Issue 6, Pages 877-885 (December 1998) PRC1  Wei Jiang, Gretchen Jimenez, Nicholas J Wells, Thomas J Hope, Geoffrey M Wahl, Tony Hunter, Rikiro Fukunaga  Molecular Cell  Volume 2, Issue 6, Pages 877-885 (December 1998) DOI: 10.1016/S1097-2765(00)80302-0

Figure 1 Identification of Human PRC1 (A) Substrate specificity of cyclin–CDK complexes against GST–PRC1 (residues 351–620) in comparison with known substrate proteins, GST–Rb (residues 768–928) and histone H1. GST–Rb, histone H1, and GST–PRC1 were incubated with purified baculovirus-expressed cyclin D1–CDK6 (K6/D1), cyclin E–CDK2 (K2/E), cyclin A–CDK2 K2/A), or cyclin B1–CDC2 (K1/B) in the presence of [γ32P]ATP. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by autoradiography (for kinase assay, top) or Coomassie blue staining (bottom). (B) Schematic domain structure of PRC1. The black box indicates a predicted α-helical domain in PRC1, and TPSK and TPGK are the consensus CDK phosphorylation sites. (C) Schematic comparison of PRC1 and Ase1p (top). The black boxes in PRC1 and Ase1p represent the regions of homology between PRC1 and Ase1p, and the open rectangles represent the putative mitotic destruction D boxes in PRC1 and the functional D box in Ase1p. Sequence of PRC1 and alignment of amino acid sequence homology regions (the black boxes in the top) of PRC1 and Ase1p using the BLAST-ψ program on the National Center for Biotechnology Information database (bottom). Amino acids that are identical are indicated by single letters between the sequence lines, amino acids that are similar are indicated by (+), and the CDK phosphorylation sites, TPSK and TPGK, are underlined. Molecular Cell 1998 2, 877-885DOI: (10.1016/S1097-2765(00)80302-0)

Figure 2 The Expression and Phosphorylation of PRC1 Protein during the Cell Cycle (A) Cell lysates from indicated cells were immunoprecipitated with anti-PRC1 antibodies or anti-PRC1 antibodies that had been prebound to PRC1 C-terminal peptide. After washing, the immunoprecipitates were subjected to SDS-PAGE, transferred to Immobilon-P, and then immunoblotted with affinity-purified anti-PRC1 antibodies. (B) HeLa cells were synchronized by a double-thymidine block and then released at different time points. Cell lysates from the indicated time points were subjected to SDS-PAGE, transferred to Immobilon-P membrane, and then blotted with affinity-purified anti-PRC1 antibodies. The PRC1 band is indicated; the asterisk represents a nonspecific band that cross-reacts weakly with anti-PRC1 antibodies. (C) Samples of the HeLa cells that were synchronized and released at different time points used in (B) were collected and analyzed for DNA content by flow cytometry (top). The values in each panel at different time points represent the percentage of cells in the indicated phase(s) of the cell cycle, determined as described in Experimental Procedures (bottom). (D) In vitro phosphorylation of GST–PRC1(wt) and its mutants (A1, A2, and A1A2) by purified, baculovirus-expressed cyclin E–CDK2. The indicated GST fusion proteins were incubated with cyclin E–CDK2 in the presence of [γ32P]ATP. Proteins were subjected to SDS-PAGE and visualized by autoradiography (for kinase assay, top) or Coomassie blue staining (bottom). (E) HeLa cells were synchronized at the indicated stages of the cell cycle and in vivo labeled with [32P]orthophosphate for a period of 4 hr. PRC1 was immunoprecipitated using affinity-purified anti-PRC1 antibodies. After washing, the immunoprecipitates were subjected to SDS-PAGE and then visualized by autoradiography. (F) Chymotrypic phosphopeptide mapping analyses of in vivo [32P]-labeled PRC1 protein obtained from mitotic cells as described in (E) and the GST–PRC1 wild type and the A1 and A2 mutants that were phosphorylated by cyclin E–CDK2 in vitro as described in (D). The in vivo and in vitro [32P]-labeled PRC1 proteins resolved by SDS-PAGE gel were eluted and then digested with α-chymotrypsin. The resulting phosphopeptides were separated by electrophoresis (pH 1.9 buffer) in the horizontal dimension (anode on the left) and chromatography (isobutyric acid buffer) in the vertical dimension. Shown are two-dimensional chymotryptic phosphopeptide maps of (wt) GST–PRC1 wild type phosphorylated by cyclin E–CDK2 in vitro (2000 cpm), (A1) A1 mutant phosphorylated by cyclin E–CDK2 in vitro (2000 cpm), (A2) A2 mutant phosphorylated by cyclin E–CDK2 in vitro (2000 cpm), the schematic map, (in vivo) PRC1 from [32P]-labeled mitotic cells (800 cpm), and (mix) mix of in vivo (400 cpm) and wt (600 cpm). The maps were exposed to phosphorimager screens for 1 day (wt, A1, and A2) and 4 days (in vivo and mix). The phosphopeptides, 1–5 and X–Z, are labeled and (*) represents the origin. Note: multiple phosphopeptides represent partial chymotryptic digestion products of a single phosphorylation site on the maps, and the altered mobility of phosphopeptide 1 on the A2 map is due to the mutation of Thr-481 to Ala. Molecular Cell 1998 2, 877-885DOI: (10.1016/S1097-2765(00)80302-0)

Figure 3 Subcellular Localization of PRC1 in HeLa Cells Asynchronous HeLa cells were grown on glass coverslips. After fixation, cells were incubated with affinity-purified rabbit polyclonal anti-PRC1 antibodies and, in parallel, with either (A) a mouse monoclonal anti-α-tubulin antibody or (B) rhodamine-conjugated phalloidin. Immunofluorescence staining was performed with FITC-conjugated goat anti-rabbit secondary antibodies and Texas red–conjugated goat anti-mouse antibodies and visualized using confocal laser scanning microscopy (CLSM). (Aa), (Ab)–(Ad), (Ae) and (Af), (Ag) and (Ah), and (Ba), (Bb), (Bc)–(Bf), and (Bg)–(Bi) represent cells in interphase, prophase or metaphase, anaphase, and telophase, respectively. (Ai) represents a newly divided daughter cell, and the arrow indicates the PRC1 staining. Scale bar is 10 μm. Molecular Cell 1998 2, 877-885DOI: (10.1016/S1097-2765(00)80302-0)

Figure 4 Effects of Microinjection of Anti-PRC1 Antibodies in HeLa Cells Asynchonously growing interphase HeLa cells were microinjected with affinity-purified anti-PRC1 antibodies (3.8 mg/ml) or control IgG (5 mg/ml). Cells were fixed, processed for immunofluorescence staining 20 hr after the injection with FITC-conjugated goat anti-rabbit secondary antibodies, and the immunofluorescence staining was analyzed using a fluorescence microscope. (A) Quantitation of the inhibition of cytokinesis in HeLa cells injected with anti-PRC1 antibodies (hatched bar) or control IgG (closed bar). The figure shows the mean results obtained from six independent experiments with error bars depicting the standard deviations. (B) HeLa cells were microinjected with anti-PRC1 antibodies as described in (A) together with fluorescein-dextran and then cultured in medium in the presence of Hoechst. Twenty hours after injection, live cells were analyzed using a fluorescence microscope. (Ba)–(Bb) represent two independent experiments. (C) Time-lapse microscopy of cells injected with anti-PRC1 antibodies described in (B). Arrow indicates the injected cell. Images are shown at 0, 30, and 60 min after the injected cell was in anaphase. Cytokinesis had failed by 30 min. Scale bar is 10 μm. Molecular Cell 1998 2, 877-885DOI: (10.1016/S1097-2765(00)80302-0)