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

Chee MK, Haase SB - PLoS Genet. (2010)

Bottom Line: 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.We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity.Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Duke University, Durham, North Carolina, United States of America.

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 SCF(Cdc4) 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 (APC(Cdh1)). 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 SCF(Cdc4) 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.

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Homology models of Kip1 and Cin8 motor domains bound to MgADP.(A) Ribbon structure overlay illustrating gross differences in the predicted structures of Kip1 (peach) and Cin8 (light blue), particularly in the length of the various loop regions. The human Eg5 (also known as Kif11) template structure (ExPDB 1ii6B) is shown in gray. The position of Ser 388 on Kip1 and Ser 455 on Cin8 is highlighted in red, while the location of the microtubule binding face and nucleotide binding pocket are indicated. (B) Close-up view of the surroundings of Ser 388 in Kip1 and Ser 455 in Cin8; both the serine residues are colored red. Residues with side chains lying within a 2.5 Å radius of the serine residue are colored light green for Kip1 and hot pink for Cin8; residues have also been labeled to highlight significant differences between the two model structures.
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pgen-1000935-g008: Homology models of Kip1 and Cin8 motor domains bound to MgADP.(A) Ribbon structure overlay illustrating gross differences in the predicted structures of Kip1 (peach) and Cin8 (light blue), particularly in the length of the various loop regions. The human Eg5 (also known as Kif11) template structure (ExPDB 1ii6B) is shown in gray. The position of Ser 388 on Kip1 and Ser 455 on Cin8 is highlighted in red, while the location of the microtubule binding face and nucleotide binding pocket are indicated. (B) Close-up view of the surroundings of Ser 388 in Kip1 and Ser 455 in Cin8; both the serine residues are colored red. Residues with side chains lying within a 2.5 Å radius of the serine residue are colored light green for Kip1 and hot pink for Cin8; residues have also been labeled to highlight significant differences between the two model structures.

Mentions: Ser 388 in Kip1 and Ser 455 in Cin8 are found in the N-terminal motor domain of the respective kinesins-5. Hence, in order to understand how phosphorylation at these residues on Kip1 and Cin8 might regulate their functions, we constructed homology models of their motor domains (Figure 8A). Homology modeling was performed using SWISS-MODEL and Swiss PDB Viewer [64]–[66] with X-ray crystal structures of the motor domains of human kinesin-5 HsEg5 [67] and the budding yeast kinesin-14 Kar3 [68] serving as templates. Consistent with the idea that phosphorylation of Ser 388 could regulate motor function, our model of the Kip1 motor domain showed that Ser 388 is solvent-accessible and located at the C-terminal end of strand β8. Here, Ser 388 appears to form part of the core which enables the motor to distinguish between ATP and ADP bound to the nucleotide-binding pocket [69]. The residue itself, however, is not predicted to form essential hydrogen bonds, and does not itself form part of the nucleotide binding pocket. Additional modeling (see Materials & Methods) showed that replacing Ser 388 with an alanine residue has no predicted effects on the backbone structure.


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

Chee MK, Haase SB - PLoS Genet. (2010)

Homology models of Kip1 and Cin8 motor domains bound to MgADP.(A) Ribbon structure overlay illustrating gross differences in the predicted structures of Kip1 (peach) and Cin8 (light blue), particularly in the length of the various loop regions. The human Eg5 (also known as Kif11) template structure (ExPDB 1ii6B) is shown in gray. The position of Ser 388 on Kip1 and Ser 455 on Cin8 is highlighted in red, while the location of the microtubule binding face and nucleotide binding pocket are indicated. (B) Close-up view of the surroundings of Ser 388 in Kip1 and Ser 455 in Cin8; both the serine residues are colored red. Residues with side chains lying within a 2.5 Å radius of the serine residue are colored light green for Kip1 and hot pink for Cin8; residues have also been labeled to highlight significant differences between the two model structures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865516&req=5

pgen-1000935-g008: Homology models of Kip1 and Cin8 motor domains bound to MgADP.(A) Ribbon structure overlay illustrating gross differences in the predicted structures of Kip1 (peach) and Cin8 (light blue), particularly in the length of the various loop regions. The human Eg5 (also known as Kif11) template structure (ExPDB 1ii6B) is shown in gray. The position of Ser 388 on Kip1 and Ser 455 on Cin8 is highlighted in red, while the location of the microtubule binding face and nucleotide binding pocket are indicated. (B) Close-up view of the surroundings of Ser 388 in Kip1 and Ser 455 in Cin8; both the serine residues are colored red. Residues with side chains lying within a 2.5 Å radius of the serine residue are colored light green for Kip1 and hot pink for Cin8; residues have also been labeled to highlight significant differences between the two model structures.
Mentions: Ser 388 in Kip1 and Ser 455 in Cin8 are found in the N-terminal motor domain of the respective kinesins-5. Hence, in order to understand how phosphorylation at these residues on Kip1 and Cin8 might regulate their functions, we constructed homology models of their motor domains (Figure 8A). Homology modeling was performed using SWISS-MODEL and Swiss PDB Viewer [64]–[66] with X-ray crystal structures of the motor domains of human kinesin-5 HsEg5 [67] and the budding yeast kinesin-14 Kar3 [68] serving as templates. Consistent with the idea that phosphorylation of Ser 388 could regulate motor function, our model of the Kip1 motor domain showed that Ser 388 is solvent-accessible and located at the C-terminal end of strand β8. Here, Ser 388 appears to form part of the core which enables the motor to distinguish between ATP and ADP bound to the nucleotide-binding pocket [69]. The residue itself, however, is not predicted to form essential hydrogen bonds, and does not itself form part of the nucleotide binding pocket. Additional modeling (see Materials & Methods) showed that replacing Ser 388 with an alanine residue has no predicted effects on the backbone structure.

Bottom Line: 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.We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity.Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Duke University, Durham, North Carolina, United States of America.

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 SCF(Cdc4) 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 (APC(Cdh1)). 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 SCF(Cdc4) 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.

Show MeSH
Related in: MedlinePlus