B-cyclin/CDKs regulate mitotic spindle assembly by phosphorylating kinesins-5 in budding yeast

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• B-cyclin/CDK complexes are crucial for mitotic spindle assembly and cell cycle progression.
• Kinesin-5 motors and SCF(Cdc4) ubiquitin ligase are essential for spindle pole separation and bipolar spindle formation.
• Previous models suggested B-cyclin/CDK complexes indirectly regulate kinesin-5 stability via APC(Cdh1).

Purpose of the Study:

• To identify novel CDK targets involved in mitotic spindle regulation.
• To elucidate the precise mechanism by which B-cyclin/CDK complexes promote spindle pole separation.
• To clarify the roles of kinesin-5 motors and SCF(Cdc4) in bipolar spindle assembly.

Main Methods:

• In vitro kinase assays to test Clb2/Cdc28 phosphorylation of Kip1 and Cin8.
• Site-directed mutagenesis of conserved CDK phosphorylation sites on Kip1.
• Analysis of spindle pole separation in wild-type and mutant yeast strains.
• Mass spectrometry to identify in vivo phosphorylation sites on Kip1.
• Investigation of SCF(Cdc4) targets involved in spindle pole separation.

Main Results:

• Kip1 and Cin8 are direct in vitro targets of Clb2/Cdc28.
• Mutation of CDK phosphorylation sites on Kip1 impairs spindle pole separation without affecting protein levels or localization.
• Mass spectrometry confirmed in vivo phosphorylation of two CDK sites in the Kip1 tail domain.
• Sic1 was identified as the SCF(Cdc4) target that inhibits spindle pole separation in cells lacking Cdc4.
• Contrary to previous suggestions, Kip1 and Cin8 levels are unaffected by Clb/Cdc28 activity in the absence of APC(Cdh1).

Conclusions:

• Clb/Cdc28 directly phosphorylates kinesin-5 motors (Kip1 and Cin8) to drive spindle pole separation.
• This phosphorylation event is a key mechanism for bipolar spindle assembly.
• Sic1 is the critical SCF(Cdc4) substrate regulating spindle pole separation when Cdc4 is absent.