Atypical regulation of a green lineage-specific B-type cyclin-dependent kinase

Abstract

Cyclin-dependent kinases (CDKs) are the main regulators of cell cycle progression in eukaryotes. The role and regulation of canonical CDKs, such as the yeast (Saccharomyces cerevisiae) Cdc2 or plant CDKA, have been extensively characterized. However, the function of the plant-specific CDKB is not as well understood. Besides being involved in cell cycle control, Arabidopsis (Arabidopsis thaliana) CDKB would integrate developmental processes to cell cycle progression. We investigated the role of CDKB in Ostreococcus (Ostreococcus tauri), a unicellular green algae with a minimal set of cell cycle genes. In this primitive alga, at the basis of the green lineage, CDKB has integrated two levels of regulations: It is regulated by Tyr phosphorylation like cdc2/CDKA and at the level of synthesis-like B-type CDKs. Furthermore, Ostreococcus CDKB/cyclin B accounts for the main peak of mitotic activity, and CDKB is able to rescue a yeast cdc28(ts) mutant. By contrast, Ostreococcus CDKA is not regulated by Tyr phosphorylation, and it exhibits a low and steady-state activity from DNA replication to exit of mitosis. This suggests that from a major role in the control of mitosis in green algae, CDKB has evolved in higher plants to assume other functions outside the cell cycle.

Figure 1.

Purification of Ostreococcus CDKs. A, Detection of Ostreococcus CDKs, with a monoclonal anti-PSTAIRE antibody, after affinity purification on CKS from either human p9 or Arabidopsis p10. B, Specificity of the anti-CDKB antibody. CDKs were purified on p10 and detected with the anti-CDKB antibody in the presence or absence of the competing antigenic peptide. C, Sequential purification of Ostreococcus CDKs by affinity chromatography. The 34- to 35-kD bands (CDKA) were depleted from an HU extract, after two rounds of affinity chromatography on p9 as detected with the anti-PSTAIRE (@PSTAIRE) antibody. Only the 37-kD CDK (CDKB) was detected on p10 beads after depletion. A band at approximately 45 kD was detected mainly in association with CDKB (p10) using an anti-cyclin B (@CycB) antibody.

Figure 2.

Expression and phosphorylation status of CDKs at different stages of cell cycle progression. Cells were arrested at different stages of the cell cycle using specific inhibitors. Olomoucine (G1s)- and HU (S-G2)-treated cells were collected 14 h after addition of the drugs. G2m and MP phase samples were obtained from artificially synchronized cells, were blocked with olomoucine or propyzamide, and were collected at the time of control mitosis exit (M/G1). Control G1 cells were collected at the time of drug addition. A, Relative frequencies of cells in G1 (white), S (gray), and G2/M (black), as detected by flow cytometry, are shown. B, CDKs were affinity purified on p10 and detected with anti-CDKB, anti-PSTAIRE, or anti-PY antibodies.

Figure 3.

Expression, cyclin partners, and activities of CDKA and CDKB at different stages of cell cycle progression. CDKA and CDKB were sequentially affinity purified (on p9 and p10, respectively) from cells arrested at different stages of cell cycle progression. A, Specific antibodies were used to detect cyclin A, cyclin B, CDKA, and CDKB bound to p9 and p10. B, Corresponding H1 kinase activities of CDKA (white) and CDKB (black) are reported (top). All activities were normalized relative to the HU sample activity (100).

Figure 4.

Expression, phosphorylation status, cyclin partners, and activities of CDKA and CDKB at different stages of cell cycle progression in cells released from an HU arrest. A, Time course analysis of DNA content of HU synchronized cells by flow cytometry. Cells were incubated with HU for 14 h and this drug was removed at time 0:00. Overlayed histographs (left section) and relative frequencies of prereplicative nuclei (G1, white circle), replicative nuclei (S, triangle), replicated nuclei (G2M, square) are as estimated by the Modfit software (right section). B, Levels, phosphorylation status, and cyclin partners of CDKA (p9 fraction) and CDKB (p10 fraction). C, Corresponding H1 kinase activities of CDKA (white; left axis) and CDKB (black; right axis).

Figure 5.

In vitro activation of Ostreococcus CDKs by Cdc25. CDKs from HU-treated cells were purified on p10, incubated with increasing amount of Cdc25, and then assayed for H1 kinase activity.

Figure 6.

Time course of CDK accumulation from G1 to an S-G2 arrest. HU was added in G1 (5 h after light on) and cells were harvested every 2 h for 13 h. A, Flow cytometry analysis of DNA content of the control population from 5 h until 18 h after light on (left) and in HU-treated cells (right). B, H1 kinase activity of CDKA (white, left axis) and CDKB (black, right axis) in HU-treated cells. C, Time course analysis and phosphorylation status of CDKA, CDKB, cyclin A, and cyclin B in the CDKA (top) and the CDKB (bottom) fractions upon accumulation from G1 to S-G2 arrest in HU-treated cells.

Figure 7.

A, Complementation studies of the budding yeast cdc28-4^ts mutant with Ostreococcus CDKA and CDKB. cdc28-4^ts mutants were transformed with either CDKB, CDC28 positive control, or no DNA (empty vector). Transformed cells were grown at permissive (25°C) or restrictive (34°C) temperature. B, Immunodetection with the anti-CDKB antibody of affinity-purified proteins on p10 from yeast cdc28-4^ts mutant expressing CDKB (lane 1) or untransformed cdc28-4^ts control cells (lane2) at permissive temperature.