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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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Kip1 and Cin8 localization in the presence and the absence of phosphorylation by Clb/Cdc28.Kip1 and Cin8 were visualized by fusion to a C-terminal mCherry tag and imaging with fluorescence microscopy. SPBs in the strains shown are marked with Spc42-GFP, and microtubules with CFP-Tub1. (A) Localization of wild-type Kip1 (top) and Cin8 (bottom), compared with that of Kip16A and Cin85A mutants, in live yeast cells. All consensus CDK sites in the mutant kinesins-5 have been mutated to non-phosphorylatable alanine. (B) Kip1 and Cin8 localization in PGAL1-CLB1 Δclb1, 2, 3, 4, 5, 6 cells 90 minutes after being released from α-factor arrest into medium containing either galactose (top panel; to induce Clb1 expression) or dextrose (bottom panel; to inhibit Clb1 expression). (C) Kip1 and Cin8 localization in wild-type cells versus cells with PGAL1-SIC1Δ3P integrated, 90 minutes after being released from α-factor arrest into galactose medium. Scale bar: 2 µm.
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pgen-1000935-g004: Kip1 and Cin8 localization in the presence and the absence of phosphorylation by Clb/Cdc28.Kip1 and Cin8 were visualized by fusion to a C-terminal mCherry tag and imaging with fluorescence microscopy. SPBs in the strains shown are marked with Spc42-GFP, and microtubules with CFP-Tub1. (A) Localization of wild-type Kip1 (top) and Cin8 (bottom), compared with that of Kip16A and Cin85A mutants, in live yeast cells. All consensus CDK sites in the mutant kinesins-5 have been mutated to non-phosphorylatable alanine. (B) Kip1 and Cin8 localization in PGAL1-CLB1 Δclb1, 2, 3, 4, 5, 6 cells 90 minutes after being released from α-factor arrest into medium containing either galactose (top panel; to induce Clb1 expression) or dextrose (bottom panel; to inhibit Clb1 expression). (C) Kip1 and Cin8 localization in wild-type cells versus cells with PGAL1-SIC1Δ3P integrated, 90 minutes after being released from α-factor arrest into galactose medium. Scale bar: 2 µm.

Mentions: To address this possibility, C-terminal mCherry [58] tags were fused to Kip1 and Cin8 expressed from their respective native promoters, and their localization was determined by fluorescence microscopy (Figure 4). We determined that Kip1-mCherry and Cin8-mCherry are both functional as they both supported growth in a kip1Δ cin8Δ background (Figure 5). We observed that wild-type Kip1 and Cin8, as well as the non-phosphorylatable mutants, Kip16A and Cin85A, localized to the spindle poles and spindle microtubules (Fig 4A). These findings suggest that phosphorylation of Kip1 and Cin8 at their consensus CDK sites is not required for spindle localization.


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

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

Kip1 and Cin8 localization in the presence and the absence of phosphorylation by Clb/Cdc28.Kip1 and Cin8 were visualized by fusion to a C-terminal mCherry tag and imaging with fluorescence microscopy. SPBs in the strains shown are marked with Spc42-GFP, and microtubules with CFP-Tub1. (A) Localization of wild-type Kip1 (top) and Cin8 (bottom), compared with that of Kip16A and Cin85A mutants, in live yeast cells. All consensus CDK sites in the mutant kinesins-5 have been mutated to non-phosphorylatable alanine. (B) Kip1 and Cin8 localization in PGAL1-CLB1 Δclb1, 2, 3, 4, 5, 6 cells 90 minutes after being released from α-factor arrest into medium containing either galactose (top panel; to induce Clb1 expression) or dextrose (bottom panel; to inhibit Clb1 expression). (C) Kip1 and Cin8 localization in wild-type cells versus cells with PGAL1-SIC1Δ3P integrated, 90 minutes after being released from α-factor arrest into galactose medium. Scale bar: 2 µm.
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Related In: Results  -  Collection

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

pgen-1000935-g004: Kip1 and Cin8 localization in the presence and the absence of phosphorylation by Clb/Cdc28.Kip1 and Cin8 were visualized by fusion to a C-terminal mCherry tag and imaging with fluorescence microscopy. SPBs in the strains shown are marked with Spc42-GFP, and microtubules with CFP-Tub1. (A) Localization of wild-type Kip1 (top) and Cin8 (bottom), compared with that of Kip16A and Cin85A mutants, in live yeast cells. All consensus CDK sites in the mutant kinesins-5 have been mutated to non-phosphorylatable alanine. (B) Kip1 and Cin8 localization in PGAL1-CLB1 Δclb1, 2, 3, 4, 5, 6 cells 90 minutes after being released from α-factor arrest into medium containing either galactose (top panel; to induce Clb1 expression) or dextrose (bottom panel; to inhibit Clb1 expression). (C) Kip1 and Cin8 localization in wild-type cells versus cells with PGAL1-SIC1Δ3P integrated, 90 minutes after being released from α-factor arrest into galactose medium. Scale bar: 2 µm.
Mentions: To address this possibility, C-terminal mCherry [58] tags were fused to Kip1 and Cin8 expressed from their respective native promoters, and their localization was determined by fluorescence microscopy (Figure 4). We determined that Kip1-mCherry and Cin8-mCherry are both functional as they both supported growth in a kip1Δ cin8Δ background (Figure 5). We observed that wild-type Kip1 and Cin8, as well as the non-phosphorylatable mutants, Kip16A and Cin85A, localized to the spindle poles and spindle microtubules (Fig 4A). These findings suggest that phosphorylation of Kip1 and Cin8 at their consensus CDK sites is not required for spindle localization.

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