The CLS domains of cyclins A and E are similar. (A) Amino acid sequence alignment for cyclin E1 and cyclin A2 CLSs. Identical residues are boxed in red and similar residues are boxed in green. (B) Ribbon diagram showing superimposition of cyclins E (yellow, PDB ID 1W98) and A (magenta, PDB ID 1VIN) CLSs in the conserved region. Identical and similar residues are represented as stick models.

The mutated cyclin A CLS DWVE-A does not displace endogenous cyclin A from centrosomes or inhibit DNA replication. (A) CHO-K1 cells were transfected with DNA constructs encoding the EGFP- cyclin A CLS domain wt or carrying CLS loss-of-function mutations (IEEK-R and DWVE-A). The vector encoding EGFP alone was used as a negative control. Centrosomal localization of endogenous cyclin A in cells expressing these constructs was monitored and compared with the localization of endogenous cyclin A in cells expressing EGFP alone. Bars indicate mean ± SEM (n = 3). (B) CHO-K1 cells were transfected with DNA constructs encoding the Myc-cyclin A CLS domain wt or carrying CLS loss-of-function mutants (IEEK-R and DWVE-A). BrdU incorporation into DNA was monitored as a marker of S phase and compared with BrdU incorporation in cells expressing a non-centrosomally-localized cyclin A fragment (aa 1–200). Black bars represent BrdU incorporation in cells expressing the Myc-construct (Myc positive cells), whereas grey bars represent BrdU incorporation in cells not expressing Myc-constructs (Myc negative cells) from the same field. Bars indicate mean ± SEM (n = 3).

Mutation of equivalent CLS residues in cyclins A and E inhibits centrosomal localization. (A) Amino acid sequence alignment of the cyclin E1 and cyclin A2 CLSs in the conserved regions. Conserved residues are boxed in blue. Residues that lead to loss of CLS function when mutated into alanine are boxed in red. In the text, the resulting mutants are named SWNQ-A for cyclin E and DWVE-A for cyclin A. (B) Xenopus S3 cells were transfected with DNA constructs encoding either EGFP-fused-cyclin A CLS wild-type (Wt) domain (aa 201–255) or with a mutant CLS domain (DWVE-A) (upper parts). Lower parts, cells transfected with EGFP-fused-full-length (Fl) cyclin A with a wild-type CLS (wt) or mutant CLS (DWVE-A). Cells were methanol-fixed and stained for γ-tubulin as a marker for centrosomes, and EGFP was monitored by direct fluorescence. Arrows indicate the position of centrosomes. Insets: magnification of the centrosomal region in the merged image. Scale bars, 10 µm.

Mutations within the cyclin A CLS reduce Cdk binding. (A) CHO-K1 cells were transfected with DNA constructs encoding EGFP-fused cyclin A Fl wt, cyclin A Fl DWVE-A or cyclin A aa 1–200 (control without a CLS). The resulting EGFP-proteins were immunoprecipitated with anti EGFP antibodies, and the efficiency of immunoprecipitation monitored by western blotting using anti-cyclin A antibodies (upper part). The binding to Cdk in the immunoprecipitate was monitored by western blot with PSTAIRE antibodies (middle part), whereas Cdk kinase activity was assayed in the immune-complex using histone H1 as a substrate (lower part, autoradiograph). (B) Ribbon diagram showing the interaction between Cdk2 (brown) and cyclin A (cyan) in the cyclin A-Cdk2 complex (PDB 1JSU). The site of the mutated residues within the cyclin A CLS region (magenta) and the major contact regions of the Cdk PSTAIRE helix and T loop are shown in red. Mutation in the cyclin A CLS, and particularly the substitution of W217 to alanine may affect the conformation of the surrounding α-helices (α3 and α4) that have multiple direct contacts (T258, I281, T282) with this residue and indirectly participate in the interaction with Cdk2 through helix α5. (C) Ribbon diagram showing the interaction between Cdk2 (brown) and cyclin E (green) in the cyclin E-Cdk2 complex (PDB 1W98). The site of the cyclin E CLS region (yellow) containing the SWNQ mutated residues, and the Cdk PSTAIRE helix and T loop are shown in red. This view highlights the proximity of the SWNQ residues to the N-helix, where cyclin E W234 contacts L229, M105 and W102, and the tip of the T loop, but does not contact the PSTAIRE helix of Cdk2.