Discovery of a distinct domain in cyclin A sufficient for centrosomal localization independently of Cdk binding

Abstract

Centrosomes have recently emerged as key regulators of the cell cycle. The G1/S transition requires a functional centrosome, and centrosomal localization of numerous proteins, including cyclin/Cdk complexes, is important for the G2/M transition. Here we identify a modular centrosomal localization signal (CLS) localizing cyclin A to centrosomes independently of Cdk binding. The cyclin A CLS is located in a distinct part of the molecule compared with the cyclin E CLS and includes the MRAIL hydrophobic patch involved in substrate recognition. The cyclin A CLS interacts with p27(KIP1), and expression of p27(KIP1) removes cyclin A but not cyclin E from centrosomes. Expression of the cyclin A CLS displaces both endogenous cyclin A and E from centrosomes and inhibits DNA replication, supporting an emerging concept that DNA replication is linked to centrosomal events. Structural analysis indicates that differences in surface charge and length of the C-terminal helix explain why the MRAIL region in cyclin E is not a functional CLS. These results indicate that the cyclin A CLS may contribute to targeting and recognition of centrosomal Cdk substrates and is required for specific effects of p27(KIP1) on cyclin A-Cdk2.

Fig. 1.

The MRAIL-containing sequence within CBOX1 localizes cyclin A to the centrosome. (A) Asynchronous CHO-K1, HeLa S3, and Xenopus S3 (XS3) cells were methanol-fixed and immunostained with antibodies to cyclin A (green) and γ-tubulin (red). (B and C) Plasmids encoding the indicated human cyclin A truncations fused to EGFP (amino acid numbers shown on the left) were transfected into XS3 cells. Cells were methanol-fixed and immunostained with antibodies to γ-tubulin. Expression and localization of the EGFP constructs were observed by direct fluorescence (green) and compared with immunolocalization of γ-tubulin (red). (D) Plasmids encoding human cyclin A truncations mutated on critical surface residues (IEEK-R) and fused to EGFP were transfected into XS3 cells. Their expression and localization were analyzed as in B. Arrows indicate the position of centrosomes. Insets: Magnification of the centrosomal region in the merged image. Line scans measuring centrosome-associated relative fluorescence intensity (rel. fluorescence intensity) are displayed on the right, with the green and red lines representing the GFP- and the γ-tubulin-associated fluorescence, respectively. (Scale bars, 10 μm.)

Fig. 6.

The CLSs of cyclins E and A are in distinct molecular regions. (A–C) Ribbon diagrams showing, respectively, the position of the cyclin E [Protein Data Bank (PDB) ID 1W98] and the cyclin A CLS (PDB ID 1VIN) within the 3D representations of the entire molecules, as well as the region of cyclin B (PDB ID 2JGZ) sufficient for centrosomal localization. The cyclin E CLS (A; yellow) belongs to the first helix of CBOX2 (231–251), whereas the cyclin A CLS (B; magenta) belongs to the first helix of CBOX1 (amino acids 210–232), and the cyclin B region sufficient for centrosomal localization (C; orange) belongs to CBOX1 and the beginning of CBOX2 (amino acids 166–311). (D and E) Space-filling models corresponding to A and B, respectively. D clearly shows that the C-terminal helix (red box) impinges on the “cyclin A CLS-like” region and may explain why this region is not a functional CLS in cyclin E. (F) Superimposition of cyclin A and E in the region containing the cyclin A CLS (magenta). The structure was rotated by ≈120° in the clockwise direction compared with Fig. 5 A–E. Residues exposed at the surface and substituted by arginines in IEEK-R mutant are annotated. This ribbon diagram also points out the differences between the C-terminal helices of cyclin A and E (bottom left corner). In cyclin E, the C-terminal helix (boxed in red) is longer than in cyclins A and B and may interfere with the accessibility of the “cyclin A CLS-like” region for protein–protein interaction. (G) Alignment of cyclin A and cyclin E CLS amino acid sequences, with the identical residues boxed in red and similar residues boxed in green.

Fig. 2.

Cyclin A centrosomal localization is independent of Cdk binding. (A) Plasmids encoding cyclin A-EGFP truncations were transfected into XS3 cells, and the resulting proteins were immunoprecipitated with an antibody to GFP (Middle). Coimmunoprecipitation of Cdk1/2 was analyzed by Western blot using an anti-PSTAIR antibody (Top). (Bottom) Histone H1 kinase activity of the cyclin A-EGFP/Cdk immune complexes (pHH1, autoradiograph). (B) Graphic representation of the different cyclin A-EGFP constructs transfected into Xenopus S3 cells and tested for their ability to localize at the centrosomes and to bind Cdk1/2. For centrosomal localization, +, ++, and +++ represent 30–50%, 50–75%, and >75% of cells with centrosomal staining, respectively. The percentage of cells with centrosomally localized cyclin A from four independent experiments is in parentheses. The stars show the position of the four mutations. Boxes indicate the cyclin A CLS.

Fig. 3.

Overexpression of the cyclin A CLS displaces endogenous cyclins A and E from centrosomes. Bar graph showing the percentage of cells with endogenous cyclins A (Left) or E (Right) localized on centrosomes in the presence of cyclin A 1–200 (dark gray), cyclin A CLS wild type (light gray), or cyclin A CLS IEEK-R (black). Bars indicate mean ± SEM (n = 4). The corresponding immunofluorescence is displayed in Fig. S2.

Fig. 4.

Overexpression of the cyclin A CLS inhibits DNA replication. Unsynchronized CHO-K1 cells were transfected with the indicated cyclin A-6myc constructs and incubated with EdU to follow DNA replication. Results are represented as a bar graph showing the percentage of cells in S-phase after transfection with the indicated constructs. For each experiment, ≈100 cells were counted. Experiments were done in triplicate. Bars indicate mean ± SEM (n = 3). The corresponding EdU fluorescence is displayed in Fig. S3A.

Fig. 5.

The Cdk inhibitor p27^KIP1 displaces cyclin A but not cyclin E from centrosomes. (A) Ribbon diagram (adapted from ref. 31) showing that the interaction between p27^KIP1 (gold) and the cyclin A-Cdk2 complex (blue and green, respectively) involves the cyclin A CLS region (purple). (B) Bar graph showing the percentage of cells with centrosomal endogenous cyclins A (black) or E (gray) after transfection with p27^KIP1-6Myc, as illustrated in C. Bars indicate mean ± SEM (n = 3). (C) CHO-K1 cells were transfected with a plasmid encoding p27^KIP1-6Myc. Cells were methanol-fixed, and expression of p27^KIP1-6Myc was assessed by indirect immunofluorescence using an anti-Myc antibody (green). Localization of endogenous cyclin A (Top) and E (Bottom) was monitored by confocal microscopy (blue) and compared with the centrosomal localization of γ-tubulin (red). Arrows indicate the position of centrosomes. Insets: Magnification of the centrosomes from the merged image. Line scans measuring centrosome-associated relative fluorescence intensity (rel. fluorescence intensity) are displayed on the right, with the green, blue, and red lines representing the 6Myc, the endogenous cyclins, and the γ-tubulin fluorescence, respectively. (Scale bars, 10 μm.)