The events of cell reproduction are governed by oscillations in the activities of cyclin-dependent kinases (Cdks). Cdks control the cell cycle by catalysing the transfer of phosphate from ATP to specific protein substrates. Despite their importance in cell-cycle control, few Cdk substrates have been identified. Here, we screened a budding yeast proteomic library for proteins that are directly phosphorylated by Cdk1 in whole-cell extracts. We identified about 200 Cdk1 substrates, several of which are phosphorylated in vivo in a Cdk1-dependent manner. The identities of these substrates reveal that Cdk1 employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events. Detailed analysis of these substrates is likely to yield important insights into cell-cycle regulation.

The fidelity of chromosome segregation depends on proper regulation of mitotic spindle behaviour. In anaphase, spindle stability is promoted by the dephosphorylation of cyclin-dependent kinase (Cdk) substrates, which results from Cdk inactivation and phosphatase activation. Few of the critical Cdk targets have been identified. Here, we identify the budding-yeast protein Fin1 (ref. 7) as a spindle-stabilizing protein whose activity is strictly limited to anaphase by changes in its phosphorylation state and rate of degradation. Phosphorylation of Fin1 from S phase to metaphase, by the cyclin-dependent kinase Clb5-Cdk1, inhibits Fin1 association with the spindle. In anaphase, when Clb5-Cdk1 is inactivated, Fin1 is dephosphorylated by the phosphatase Cdc14. Fin1 dephosphorylation targets it to the poles and microtubules of the elongating spindle, where it contributes to spindle integrity. A non-phosphorylatable Fin1 mutant localizes to the spindle before anaphase and impairs efficient chromosome segregation. As cells complete mitosis and disassemble the spindle, the ubiqutin ligase APC(Cdh1) targets Fin1 for destruction. Our studies illustrate how phosphorylation-dependent changes in the behaviour of Cdk1 substrates influence complex mitotic processes.

Stabilization of spindle microtubules during anaphase is essential for proper chromosome segregation. Fin1 is a budding yeast protein that localizes to the poles and microtubules of the spindle during anaphase and contributes to spindle stability. The N-terminal half of Fin1 is phosphorylated at multiple sites by the cyclin-dependent kinase Clb5-Cdk1, and dephosphorylation in anaphase triggers its localization to the spindle. The C-terminal half of Fin1 contains coiled-coil motifs that are required for its self-association. Here we investigated the functional importance of the two regions of Fin1. Fin1 mutants lacking the C-terminal coiled-coil domains localized to spindle pole bodies but not along spindle microtubules. These mutants failed to self-associate and displayed reduced binding to microtubules in vitro but were functional in vivo and stabilized anaphase spindles when dephosphorylated. Deletion of the Fin1 C terminus suppressed the lethal phenotypes of the phospho-mutant Fin1 5A . Our findings suggest that the N-terminal region of Fin1 is sufficient for its regulated function as a spindle-stabilizing factor and that this function involves association with the spindle pole body. The ability of the C-terminal region to promote Fin1 self-association and microtubule binding may underlie the lethal effects of the deregulated Fin1 5A mutant.The faithful segregation of chromosomes during mitosis requires precise regulatory control of spindle microtubule dynamics. During metaphase, microtubules exhibit high dynamic instability, which facilitates the capture of chromosomes at their kinetochores. At the onset of anaphase, microtubule dynamics are silenced, and the spindle is thereby stabilized (1-3). The change in microtubule dynamics at the onset of anaphase depends on regulated changes in the activity of proteins that influence microtubule behavior (4).The budding yeast protein Fin1 is a microtubule-stabilizing factor that associates with the spindle during anaphase. The activity of Fin1 is carefully regulated during the cell cycle (5). Phosphorylation in early mitosis by the cyclin-dependent kinase Clb5-Cdk1 inhibits Fin1 function and prevents its association with the spindle. Dephosphorylation in anaphase promotes its localization to the spindle, where it acts as a stability factor. A Fin1 phospho-mutant (Fin1 5A ) localizes prematurely to the spindle, alters metaphase spindle structure, and causes lethal errors in chromosome segregation (5).Fin1 purified from yeast self-associates to form a 10-nm-diameter filament (6). Fin1-Fin1 interactions have also been demonstrated in vivo (7,8). Oligomerization of Fin1 is mediated by sequences in the C-terminal portion of the protein, which are predicted to form coiled-coils (9). It has been suggested that Fin1 filaments provide structural support to the mitotic spindle, but the importance of Fin1 self-association for spindle stability is unknown (10, 11).We tested the importance of Fin1 self-association by analyzing the properties of a series of Fin1 truncations. We fo...