Limits...
Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation.

Toyo-oka K, Mori D, Yano Y, Shiota M, Iwao H, Goto H, Inagaki M, Hiraiwa N, Muramatsu M, Wynshaw-Boris A, Yoshiki A, Hirotsune S - J. Cell Biol. (2008)

Bottom Line: Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1.Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition.Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Disease Research and 2Department of Pharmacology, Osaka City University Graduate School of Medicine, Osaka 545-8586, Japan.

ABSTRACT
Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.

Show MeSH

Related in: MedlinePlus

Rescue experiments in PP4c-disrupted MEF cells by a Cdk1 inhibitor, additional disruption of Ndel1, and siRNA against katanin p60. (A) Depletion of Cdk1 by siRNA rescued the defect of MTs in PP4c−/− MEF cells (type A). (left) MT array was rescued by the depletion of Cdk1. (middle) T219 phosphorylation of NDEL1 (left) and katanin p60 distribution (right) under control siRNA or Cdk1 siRNA in PP4c−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of Cdk1 inhibition on MT defects (n = 200 for each of PP4ccko/cko; control siRNA, PP4ccko/cko; Cdk1 siRNA, PP4c−/−; control siRNA, and PP4c−/−; Cdk1 siRNA). (B) Ndel1 deletion can rescue the defect of MTs in PP4c−/− MEF cells (type A). (left) PP4c−/−/Ndel1−/− MEF cells show a normal MT array compared with PP4c−/−/Ndel1+/+ MEF cells. (middle) S123 phosphorylation of cyclin B1 (left) and katanin p60 distribution (right) in PP4ccko/cko/Ndel1cko/cko MEF cells (RFP-Cre negative) or PP4c−/−/Ndel1−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of Ndel1 deletion on MT abnormality (n = 100 for each of PP4ccko/cko/Ndel1cko/cko, PP4c−/−/Ndel1+/+, PP4c+/+/Ndel1−/−, and PP4c−/−/Ndel1−/−). (C) Rescue experiments with siRNA against katanin p60 in PP4c−/− MEF cells (type A). (left) Depletion of katanin p60–rescued MT defects in PP4c−/− MEF cells. (middle) Depletion of katanin p60 did not prevent the aberrant NDEL1 phosphorylation (left) and the unscheduled cyclin B1 phosphorylation (right) in PP4c−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of katanin p60 depletion by siRNA against katanin p60 on MT defects (n = 200 each of Cre−/ctrlRNAi+, CRE+/ctrlRNAi+, CRE−/p60RNAi+, and CRE+/p60RNAi+). (A–C) Arrowheads indicate the positions of centrosomes. Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2290842&req=5

fig7: Rescue experiments in PP4c-disrupted MEF cells by a Cdk1 inhibitor, additional disruption of Ndel1, and siRNA against katanin p60. (A) Depletion of Cdk1 by siRNA rescued the defect of MTs in PP4c−/− MEF cells (type A). (left) MT array was rescued by the depletion of Cdk1. (middle) T219 phosphorylation of NDEL1 (left) and katanin p60 distribution (right) under control siRNA or Cdk1 siRNA in PP4c−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of Cdk1 inhibition on MT defects (n = 200 for each of PP4ccko/cko; control siRNA, PP4ccko/cko; Cdk1 siRNA, PP4c−/−; control siRNA, and PP4c−/−; Cdk1 siRNA). (B) Ndel1 deletion can rescue the defect of MTs in PP4c−/− MEF cells (type A). (left) PP4c−/−/Ndel1−/− MEF cells show a normal MT array compared with PP4c−/−/Ndel1+/+ MEF cells. (middle) S123 phosphorylation of cyclin B1 (left) and katanin p60 distribution (right) in PP4ccko/cko/Ndel1cko/cko MEF cells (RFP-Cre negative) or PP4c−/−/Ndel1−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of Ndel1 deletion on MT abnormality (n = 100 for each of PP4ccko/cko/Ndel1cko/cko, PP4c−/−/Ndel1+/+, PP4c+/+/Ndel1−/−, and PP4c−/−/Ndel1−/−). (C) Rescue experiments with siRNA against katanin p60 in PP4c−/− MEF cells (type A). (left) Depletion of katanin p60–rescued MT defects in PP4c−/− MEF cells. (middle) Depletion of katanin p60 did not prevent the aberrant NDEL1 phosphorylation (left) and the unscheduled cyclin B1 phosphorylation (right) in PP4c−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of katanin p60 depletion by siRNA against katanin p60 on MT defects (n = 200 each of Cre−/ctrlRNAi+, CRE+/ctrlRNAi+, CRE−/p60RNAi+, and CRE+/p60RNAi+). (A–C) Arrowheads indicate the positions of centrosomes. Bars, 20 μm.

Mentions: First, to explore the causative relationships between the unscheduled activation of Cdk1 and disorganization of MTs, we used an siRNA knockdown approach to inactivate Cdk1 (Fig. 7 A and Fig. S5 A, available at http://www.jcb.org/cgi/content/full/jcb.200705148/DC1). Silencing of Cdk1 clearly suppressed the phosphorylation of NDEL1 at T219, which lead to the reduction of katanin p60 recruitment to the centrosome, resulting in an improvement of MT organization. As another method to inhibit Cdk1 activity, PP4c−/− MEF cells were cultured in the presence of the Cdk inhibitor butyrolactone I (Fig. S5 B; Kitagawa et al., 1993). Inhibition of Cdk1 suppressed the phosphorylation of NDEL1 at T219 and facilitated the redistribution of katanin p60 from the centrosome to the cytoplasm, resulting in a reduction of katanin p60 concentration at the centrosome. As with Cdk1 siRNA, butyrolactone I resulted in an improvement of the MT organization in PP4c−/− MEF cells (Fig. S5 B). These observations suggest that abnormal activation of Cdk1 may be a causative mechanism for abnormal katanin p60 accumulation at the centrosome.


Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation.

Toyo-oka K, Mori D, Yano Y, Shiota M, Iwao H, Goto H, Inagaki M, Hiraiwa N, Muramatsu M, Wynshaw-Boris A, Yoshiki A, Hirotsune S - J. Cell Biol. (2008)

Rescue experiments in PP4c-disrupted MEF cells by a Cdk1 inhibitor, additional disruption of Ndel1, and siRNA against katanin p60. (A) Depletion of Cdk1 by siRNA rescued the defect of MTs in PP4c−/− MEF cells (type A). (left) MT array was rescued by the depletion of Cdk1. (middle) T219 phosphorylation of NDEL1 (left) and katanin p60 distribution (right) under control siRNA or Cdk1 siRNA in PP4c−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of Cdk1 inhibition on MT defects (n = 200 for each of PP4ccko/cko; control siRNA, PP4ccko/cko; Cdk1 siRNA, PP4c−/−; control siRNA, and PP4c−/−; Cdk1 siRNA). (B) Ndel1 deletion can rescue the defect of MTs in PP4c−/− MEF cells (type A). (left) PP4c−/−/Ndel1−/− MEF cells show a normal MT array compared with PP4c−/−/Ndel1+/+ MEF cells. (middle) S123 phosphorylation of cyclin B1 (left) and katanin p60 distribution (right) in PP4ccko/cko/Ndel1cko/cko MEF cells (RFP-Cre negative) or PP4c−/−/Ndel1−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of Ndel1 deletion on MT abnormality (n = 100 for each of PP4ccko/cko/Ndel1cko/cko, PP4c−/−/Ndel1+/+, PP4c+/+/Ndel1−/−, and PP4c−/−/Ndel1−/−). (C) Rescue experiments with siRNA against katanin p60 in PP4c−/− MEF cells (type A). (left) Depletion of katanin p60–rescued MT defects in PP4c−/− MEF cells. (middle) Depletion of katanin p60 did not prevent the aberrant NDEL1 phosphorylation (left) and the unscheduled cyclin B1 phosphorylation (right) in PP4c−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of katanin p60 depletion by siRNA against katanin p60 on MT defects (n = 200 each of Cre−/ctrlRNAi+, CRE+/ctrlRNAi+, CRE−/p60RNAi+, and CRE+/p60RNAi+). (A–C) Arrowheads indicate the positions of centrosomes. Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2290842&req=5

fig7: Rescue experiments in PP4c-disrupted MEF cells by a Cdk1 inhibitor, additional disruption of Ndel1, and siRNA against katanin p60. (A) Depletion of Cdk1 by siRNA rescued the defect of MTs in PP4c−/− MEF cells (type A). (left) MT array was rescued by the depletion of Cdk1. (middle) T219 phosphorylation of NDEL1 (left) and katanin p60 distribution (right) under control siRNA or Cdk1 siRNA in PP4c−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of Cdk1 inhibition on MT defects (n = 200 for each of PP4ccko/cko; control siRNA, PP4ccko/cko; Cdk1 siRNA, PP4c−/−; control siRNA, and PP4c−/−; Cdk1 siRNA). (B) Ndel1 deletion can rescue the defect of MTs in PP4c−/− MEF cells (type A). (left) PP4c−/−/Ndel1−/− MEF cells show a normal MT array compared with PP4c−/−/Ndel1+/+ MEF cells. (middle) S123 phosphorylation of cyclin B1 (left) and katanin p60 distribution (right) in PP4ccko/cko/Ndel1cko/cko MEF cells (RFP-Cre negative) or PP4c−/−/Ndel1−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of Ndel1 deletion on MT abnormality (n = 100 for each of PP4ccko/cko/Ndel1cko/cko, PP4c−/−/Ndel1+/+, PP4c+/+/Ndel1−/−, and PP4c−/−/Ndel1−/−). (C) Rescue experiments with siRNA against katanin p60 in PP4c−/− MEF cells (type A). (left) Depletion of katanin p60–rescued MT defects in PP4c−/− MEF cells. (middle) Depletion of katanin p60 did not prevent the aberrant NDEL1 phosphorylation (left) and the unscheduled cyclin B1 phosphorylation (right) in PP4c−/− MEF cells (RFP-Cre positive). One example of three independent experiments is shown. (right) Statistical analysis of the effect of katanin p60 depletion by siRNA against katanin p60 on MT defects (n = 200 each of Cre−/ctrlRNAi+, CRE+/ctrlRNAi+, CRE−/p60RNAi+, and CRE+/p60RNAi+). (A–C) Arrowheads indicate the positions of centrosomes. Bars, 20 μm.
Mentions: First, to explore the causative relationships between the unscheduled activation of Cdk1 and disorganization of MTs, we used an siRNA knockdown approach to inactivate Cdk1 (Fig. 7 A and Fig. S5 A, available at http://www.jcb.org/cgi/content/full/jcb.200705148/DC1). Silencing of Cdk1 clearly suppressed the phosphorylation of NDEL1 at T219, which lead to the reduction of katanin p60 recruitment to the centrosome, resulting in an improvement of MT organization. As another method to inhibit Cdk1 activity, PP4c−/− MEF cells were cultured in the presence of the Cdk inhibitor butyrolactone I (Fig. S5 B; Kitagawa et al., 1993). Inhibition of Cdk1 suppressed the phosphorylation of NDEL1 at T219 and facilitated the redistribution of katanin p60 from the centrosome to the cytoplasm, resulting in a reduction of katanin p60 concentration at the centrosome. As with Cdk1 siRNA, butyrolactone I resulted in an improvement of the MT organization in PP4c−/− MEF cells (Fig. S5 B). These observations suggest that abnormal activation of Cdk1 may be a causative mechanism for abnormal katanin p60 accumulation at the centrosome.

Bottom Line: Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1.Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition.Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Disease Research and 2Department of Pharmacology, Osaka City University Graduate School of Medicine, Osaka 545-8586, Japan.

ABSTRACT
Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.

Show MeSH
Related in: MedlinePlus