Limits...
Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.

Robbins JA, Cross FR - Mol. Biol. Cell (2010)

Bottom Line: Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation.Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1.Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10065, USA.

ABSTRACT
Anaphase promoting complex (APC)-Cdh1 targets multiple mitotic proteins for degradation upon exit from mitosis into G1; inhibitory phosphorylation of Cdh1 by cyclin-dependent kinase (CDK) and Polo kinase has been proposed to prevent the premature degradation of substrates in the ensuing cell cycle. Here, we demonstrate essentiality of CDK phosphorylation of Cdh1 in Saccharomyces cerevisiae by exact endogenous gene replacement of CDH1 with CDK-unphosphorylatable CDH1-m11; in contrast, neither Cdh1 polo kinase sites nor polo interaction motifs are required. CDH1-m11 cells arrest in the first cycle with replicated DNA and sustained polarized growth; most cells have monopolar spindles. Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation. In contrast, expression of undegradable mitotic cyclin results in both SPB separation and the restoration of isotropic growth. A minority of CDH1-m11 cells arrest with short bipolar spindles that fail to progress to anaphase; this can be accounted for by a failure to accumulate Cdc20 and consequent failure to cleave cohesin. Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

Show MeSH

Related in: MedlinePlus

CDH1-m11 cells accumulate Pds1 but not Cdc20, and lengthen their spindles upon cohesin inactivation. (A) Left, immunoblots against strains synchronously released from α-factor with endogenously tagged Cdc20 and either CDH1 or CDH1-m11. Right, quantification of normalized Myc-Cdc20 levels from immunoblots. (B) Left, immunoblots against strains synchronously released from α-factor with endogenously tagged Pds1 and either CDH1 or CDH1-m11. Right, quantification of Myc-Pds1 levels standardized to Pgk1 loading control. (C) Average distance between separated SPBs in CDH1-m11 SCC1 and temperature-sensitive CDH1-m11 scc1-73 cells, 2 h after release from α-factor block. (D) Histogram of distance between SPBs from C.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2836972&req=5

Figure 5: CDH1-m11 cells accumulate Pds1 but not Cdc20, and lengthen their spindles upon cohesin inactivation. (A) Left, immunoblots against strains synchronously released from α-factor with endogenously tagged Cdc20 and either CDH1 or CDH1-m11. Right, quantification of normalized Myc-Cdc20 levels from immunoblots. (B) Left, immunoblots against strains synchronously released from α-factor with endogenously tagged Pds1 and either CDH1 or CDH1-m11. Right, quantification of Myc-Pds1 levels standardized to Pgk1 loading control. (C) Average distance between separated SPBs in CDH1-m11 SCC1 and temperature-sensitive CDH1-m11 scc1-73 cells, 2 h after release from α-factor block. (D) Histogram of distance between SPBs from C.

Mentions: A failure of cohesin cleavage not dependent upon checkpoint activation could also explain failure of anaphase. Cdh1 may target Cdc20 for degradation (Huang et al., 2001). Cdc20 promotes anaphase by degradation of the separase inhibitor Pds1, allowing cleavage of the cohesin complex subunit Scc1; sister chromatids can then separate upon loss of cohesion. Failure to accumulate sufficient Cdc20, if it results in an inability to clear Pds1, could account for persistent short bipolar spindles. We find that CDH1-m11 cells fail to accumulate Cdc20 (Figure 5A).


Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.

Robbins JA, Cross FR - Mol. Biol. Cell (2010)

CDH1-m11 cells accumulate Pds1 but not Cdc20, and lengthen their spindles upon cohesin inactivation. (A) Left, immunoblots against strains synchronously released from α-factor with endogenously tagged Cdc20 and either CDH1 or CDH1-m11. Right, quantification of normalized Myc-Cdc20 levels from immunoblots. (B) Left, immunoblots against strains synchronously released from α-factor with endogenously tagged Pds1 and either CDH1 or CDH1-m11. Right, quantification of Myc-Pds1 levels standardized to Pgk1 loading control. (C) Average distance between separated SPBs in CDH1-m11 SCC1 and temperature-sensitive CDH1-m11 scc1-73 cells, 2 h after release from α-factor block. (D) Histogram of distance between SPBs from C.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2836972&req=5

Figure 5: CDH1-m11 cells accumulate Pds1 but not Cdc20, and lengthen their spindles upon cohesin inactivation. (A) Left, immunoblots against strains synchronously released from α-factor with endogenously tagged Cdc20 and either CDH1 or CDH1-m11. Right, quantification of normalized Myc-Cdc20 levels from immunoblots. (B) Left, immunoblots against strains synchronously released from α-factor with endogenously tagged Pds1 and either CDH1 or CDH1-m11. Right, quantification of Myc-Pds1 levels standardized to Pgk1 loading control. (C) Average distance between separated SPBs in CDH1-m11 SCC1 and temperature-sensitive CDH1-m11 scc1-73 cells, 2 h after release from α-factor block. (D) Histogram of distance between SPBs from C.
Mentions: A failure of cohesin cleavage not dependent upon checkpoint activation could also explain failure of anaphase. Cdh1 may target Cdc20 for degradation (Huang et al., 2001). Cdc20 promotes anaphase by degradation of the separase inhibitor Pds1, allowing cleavage of the cohesin complex subunit Scc1; sister chromatids can then separate upon loss of cohesion. Failure to accumulate sufficient Cdc20, if it results in an inability to clear Pds1, could account for persistent short bipolar spindles. We find that CDH1-m11 cells fail to accumulate Cdc20 (Figure 5A).

Bottom Line: Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation.Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1.Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

View Article: PubMed Central - PubMed

Affiliation: The Rockefeller University, New York, NY 10065, USA.

ABSTRACT
Anaphase promoting complex (APC)-Cdh1 targets multiple mitotic proteins for degradation upon exit from mitosis into G1; inhibitory phosphorylation of Cdh1 by cyclin-dependent kinase (CDK) and Polo kinase has been proposed to prevent the premature degradation of substrates in the ensuing cell cycle. Here, we demonstrate essentiality of CDK phosphorylation of Cdh1 in Saccharomyces cerevisiae by exact endogenous gene replacement of CDH1 with CDK-unphosphorylatable CDH1-m11; in contrast, neither Cdh1 polo kinase sites nor polo interaction motifs are required. CDH1-m11 cells arrest in the first cycle with replicated DNA and sustained polarized growth; most cells have monopolar spindles. Blocking proteolysis of the Cin8 kinesin in CDH1-m11 cells does not promote spindle pole body (SPB) separation. In contrast, expression of undegradable mitotic cyclin results in both SPB separation and the restoration of isotropic growth. A minority of CDH1-m11 cells arrest with short bipolar spindles that fail to progress to anaphase; this can be accounted for by a failure to accumulate Cdc20 and consequent failure to cleave cohesin. Bipolar spindle assembly in CDH1-m11 cells is strikingly sensitive to gene dosage of the stoichiometric Cdh1 inhibitor ACM1. Thus, different spindle-regulatory pathways have distinct sensitivities to Cdh1, and ACM1 may buffer essential CDK phosphorylation of Cdh1.

Show MeSH
Related in: MedlinePlus