Cyclin A2 mutagenesis analysis: a new insight into CDK activation and cellular localization requirements

Abstract

Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved.

Figure 1. Schematic representation of mouse Cyclin A2 cDNA with the modifications introduced in this study.

All constructs were C-terminally fused to 3 Flag tags.

Figure 2. Analysis of the binding and kinase-activating properties of different Cyclin A2 alleles in NIH3T3 cells.

WT, D171A, Triple (M200A, L204A and W207A), M200A, L204A, W207A (A), and WT, E180A, E210Q, Double (W207L, E210Q), K256A, L282R and E285A Cyclin A2 alleles (B). Immunoprecipitation using anti-Flag-agarose affinity gel was performed on NIH3T3 cells extracts after transfection of plasmids encoding the above-mentioned mutants. Immune complexes were either submitted to Western blotting to identify binding partners or analyzed for their histone H1-kinase activity. Schematic representation of the importance of the different Cyclin A2 domains for partners association and kinase-induced activity. The bars represent (high-medium-low according to their size) the importance of the indicated region of Cyclin A2 for its association with partners or kinase inducing activity (C).

Figure 3. Analysis of binding and kinase-activating properties of different Cyclin A2 alleles in HEK 293 cells.

WT, Triple (M200A, L204A, W207A), D171A (A) and M200A, L204A and W207A Cyclin A2 alleles (B). Immunoprecipitation using anti-Flag-agarose affinity gel was performed on HEK 293 cells extracts after transfection of vectors encoding the above-mentioned mutants. Immune complexes were either submitted to Western blotting to identify binding partners or analyzed for their histone H1-kinase activity.

Figure 4. Immunofluorescence analysis of cellular localization for different Cyclin A2 alleles in NIH3T3 cells.

WT, L204A, W207A, Triple (M200A, L204A, W207A), E180A, E210Q, Double (W207L, E210Q), K256A, L282R and E285A Cyclin A2 alleles. Staining performed using anti-Flag antibody (green) and Hoechst (blue) in NIH3T3 cells transfected with vectors encoding these Cyclin A2 alleles. Scale bar: 20 µm.

Figure 5. Immunofluorescent analysis of Cyclin A2 localization in p21 ^−/−, p107 ^−/−, p27 ^−/−, CDK2 ^−/− and double p27 ^−/− CDK2 ^−/− genetic backgrounds.

Staining was performed using anti-Cyclin A2 antibody (green) for endogenous Cyclin A2. Hoechst (blue) was used for nuclear staining. Scale bar: 20 µm.

Figure 6. Immunofluorescence analysis of localization for Cyclin A2 deletion mutants.

Immunofluorescent staining for Cyclin A2 deletion mutants localization using anti-Flag antibody (A). Schematic representation of Cyclin A2 deletion mutants (B). Western blot analysis of Cyclin A2 deletion mutants expression using anti-Flag antibody (C). Scale bar: 20 µm.