B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast

Mark K Chee,Steven B Haase,Sue Biggins

doi:10.1371/journal.pgen.1000935

Abstract

Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.

Highlights

Cyclin-dependent kinases (CDKs) complexed with various cyclins coordinate many duplication and segregation events during the eukaryotic cell division cycle [1,2]
Previous genetic studies indicate that kinesin-5 motors, Clb/ Cdc28 complexes, and the SCF complex are all required for spindle pole body (SPB) separation and assembly of a mitotic spindle
We have shown that the only important target for SCFCdc4 in SPB separation is the Clb-specific CDK inhibitor, Sic1 (Figure 1)

Summary

Introduction

Cyclin-dependent kinases (CDKs) complexed with various cyclins coordinate many duplication and segregation events during the eukaryotic cell division cycle [1,2]. Separation of the duplicated centrosomes is required for the assembly of the bipolar spindle at metaphase which, in turn, is necessary for the equal segregation of sister chromatids during anaphase and the preservation of genome stability. The budding yeast centrosome, called the spindle pole body (SPB), is functionally equivalent to the metazoan centrosome. The budding yeast SPB is a powerful model for understanding the metazoan centrosome, as demonstrated by genetic studies that have identified many components of the eukaryotic cellular machinery critical to both SPB and centrosome separation (reviewed in [5,6,7,8,9])

Methods

Results

Conclusion