The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores

eLife ◽

10.7554/elife.12902 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 45

Author(s):

Sushama Sivakumar ◽

Paweł Ł Janczyk ◽

Qianhui Qu ◽

Chad A Brautigam ◽

P Todd Stukenberg ◽

...

Keyword(s):

Protein Phosphatase ◽

Force Production ◽

Dominant Negative ◽

Protein Phosphatase 1 ◽

Negative Effects ◽

Checkpoint Signaling ◽

Anaphase Onset ◽

Spindle Microtubules ◽

Cell Cycle Transition

The spindle- and kinetochore-associated (Ska) complex is essential for normal anaphase onset in mitosis. The C-terminal domain (CTD) of Ska1 binds microtubules and was proposed to facilitate kinetochore movement on depolymerizing spindle microtubules. Here, we show that Ska complex recruits protein phosphatase 1 (PP1) to kinetochores. This recruitment requires the Ska1 CTD, which binds PP1 in vitro and in human HeLa cells. Ska1 lacking its CTD fused to a PP1-binding peptide or fused directly to PP1 rescues mitotic defects caused by Ska1 depletion. Ska1 fusion to catalytically dead PP1 mutant does not rescue and shows dominant negative effects. Thus, the Ska complex, specifically the Ska1 CTD, recruits PP1 to kinetochores to oppose spindle checkpoint signaling kinases and promote anaphase onset. Microtubule binding by Ska, rather than acting in force production for chromosome movement, may instead serve to promote PP1 recruitment to kinetochores fully attached to spindle microtubules at metaphase.

Download Full-text

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

eLife ◽

10.7554/elife.25366 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 34

Author(s):

Margarida Moura ◽

Mariana Osswald ◽

Nelson Leça ◽

João Barbosa ◽

António J Pereira ◽

...

Keyword(s):

Protein Phosphatase ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Exit ◽

Protein Phosphatase 1 ◽

Anaphase Onset ◽

Phosphorylation Cascade ◽

Spindle Microtubules ◽

Early Mitosis

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Download Full-text

Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation

The Journal of Cell Biology ◽

10.1083/jcb.201909136 ◽

2020 ◽

Vol 219 (4) ◽

Cited By ~ 2

Author(s):

Gisela Cairo ◽

Anne M. MacKenzie ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Protein Phosphatase ◽

Mitotic Chromosome ◽

Budding Yeast ◽

Meiotic Chromosome ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Spindle Microtubules

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore–microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1–Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome–spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1–Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

Download Full-text

Nuclear organisation of NIPP1, a regulatory subunit of protein phosphatase 1 that associates with pre-mRNA splicing factors

Journal of Cell Science ◽

10.1242/jcs.112.2.157 ◽

1999 ◽

Vol 112 (2) ◽

pp. 157-168 ◽

Cited By ~ 12

Author(s):

L. Trinkle-Mulcahy ◽

P. Ajuh ◽

A. Prescott ◽

F. Claverie-Martin ◽

S. Cohen ◽

...

Keyword(s):

Protein Phosphatase ◽

Mrna Splicing ◽

Dominant Negative ◽

Protein Phosphatase 1 ◽

Regulatory Subunit ◽

Dominant Negative Mutant ◽

Splicing Factors ◽

Nuclear Extracts ◽

Nuclear Organisation

Protein phosphatase-1 (PP1) is complexed to many proteins that target it to particular subcellular locations and regulate its activity. Here, we show that ‘nuclear inhibitor of PP1’ (NIPP1), a major nuclear PP1-binding protein, shows a speckled nucleoplasmic distribution where it is colocalised with pre-mRNA splicing factors. One of these factors (Sm) is also shown to be complexed to NIPP1 in nuclear extracts. Immunodepletion of NIPP1 from nuclear extracts, or addition of a ‘dominant negative’ mutant lacking a functional PP1 binding site, greatly reduces pre-mRNA splicing activity in vitro. These findings implicate the NIPP1-PP1 complex in the control of pre-mRNA splicing.

Download Full-text

Bub3 and Bub1 maintain the balance of kinetochore-localized Aurora B Kinase and Protein Phosphatase I to Regulate Chromosome Segregation and Anaphase Onset in Meiosis

10.1101/778480 ◽

2019 ◽

Cited By ~ 1

Author(s):

Gisela Cairo ◽

Anne M. MacKenzie ◽

Soni Lacefield

Keyword(s):

Chromosome Segregation ◽

Protein Phosphatase ◽

Meiotic Chromosome ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Aurora B Kinase ◽

Checkpoint Proteins ◽

Anaphase Onset ◽

Normal Duration ◽

Spindle Microtubules

AbstractAccurate chromosome segregation depends on proper attachment of kinetochores to spindle microtubules prior to anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore-microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1/Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1, and concomitant massive chromosome mis-segregation caused by incorrect chromosome-spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1-Bub3 pathway in maintaining the balance between kinetochore-localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.SummaryCairo et al show that in S. cerevisiae meiosis, spindle checkpoint proteins Bub1 and Bub3 have an essential role in preventing chromosome mis-segregation and setting the normal duration of anaphase I and anaphase II onset by regulating the kinetochore-localization of Ipl1 and PP1.

Download Full-text

Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽

10.1182/blood.v93.12.4154 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4154-4166 ◽

Cited By ~ 80

Author(s):

Robert L. Ilaria ◽

Robert G. Hawley ◽

Richard A. Van Etten

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Cytokine Signaling ◽

Transactivation Domain ◽

Negative Effects ◽

Murine Bone Marrow ◽

Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

Download Full-text

Abstract 70: Protein Phosphatase 1 Contributes to Atrial Stunning in Atrial Fibrillation

Circulation Research ◽

10.1161/res.121.suppl_1.70 ◽

2017 ◽

Vol 121 (suppl_1) ◽

Author(s):

Srikanth Perike ◽

Xander Wehrens ◽

Dawood Darbar ◽

Mark McCauley

Keyword(s):

Atrial Fibrillation ◽

Light Chain ◽

Protein Phosphatase ◽

Myosin Light Chain ◽

Stroke Risk ◽

Protein Phosphatase 1 ◽

Regulatory Subunit ◽

Future Studies ◽

Hek Cells

Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia, and increases a patient’s stroke risk five-fold. Reduced atrial contractility (stunning) is observed in AF and contributes to stroke risk; however, the mechanisms responsible for atrial stunning in AF are unknown. Recent data from our laboratory indicate that protein phosphatase 1 (PP1) dephosphorylation of myosin light chain 2a (MLC2a) may contribute to atrial stunning in AF. Objective: To determine how the PP1 regulatory subunit 12C (PPP1R12C) and catalytic (PPP1c) subunits modify atrial sarcomere phosphorylation in AF. Methods: We evaluated the protein expression, binding and phosphorylation among PPP1R12C, PPP1c, and MLC2a in transfected HL-1 cells, murine atrial tissue (Pitx2null +/– mice, with a genetic predisposition AF), and in HEK cells. An inhibitor of PPP1R12C phosphorylation, BDP5290, was used to enhance the PPP1R12C-PPP1C interaction. Results: In Pitx2 null +/– mice, PPP1R12C was increased by 2-fold ( P <0.01) and associated with a 40% reduction in S-19-MLC2a phosphorylation versus WT mice ( P <0.058). BDP5290 increased PPP1R12C-PPP1C binding by >3-fold in HL-1 cells ( P <0.01). BDP5290 reduced MLC2a phosphorylation by 40% through an enhanced interaction with PPP1R12C by >3-fold in HEK cells ( P <0.01). Conclusion: In Pitx2 null+/- mice, increased expression of PPP1R12C is associated with PP1 holoenzyme targeting to sarcomeric MLC2a, and is associated with reduced S19-MLC2a phosphorylation. Additionally, BDP5290 enhances the PPP1R12C-PPP1C interaction and models PP1 activity in AF. Future studies will examine the effects of both AF and BDP5290 upon atrial contractility in vitro.

Download Full-text

The Ki-67 and RepoMan mitotic phosphatases assemble via an identical, yet novel mechanism

eLife ◽

10.7554/elife.16539 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 31

Author(s):

Ganesan Senthil Kumar ◽

Ezgi Gokhan ◽

Sofie De Munter ◽

Mathieu Bollen ◽

Paola Vagnarelli ◽

...

Keyword(s):

Protein Phosphatase ◽

Mitotic Chromosome ◽

Cancer Therapeutics ◽

Mitotic Exit ◽

Protein Phosphatase 1 ◽

Aurora B ◽

Aurora B Kinase ◽

Ki 67 ◽

Anaphase Onset

Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (<xref ref-type="bibr" rid="bib2">Booth et al., 2014</xref>). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.

Download Full-text

Protein phosphatase 1 binds to phospho-Ser-1394 of the macrophage-stimulating protein receptor

Biochemical Journal ◽

10.1042/bj20030391 ◽

2003 ◽

Vol 376 (3) ◽

pp. 587-594 ◽

Cited By ~ 5

Author(s):

Massimo M. SANTORO ◽

Giovanni GAUDINO ◽

Emma VILLA-MORUZZI

Keyword(s):

Protein Phosphatase ◽

Growth Factor Receptor ◽

Living Cells ◽

Protein Phosphatase 1 ◽

Serine Threonine Kinase ◽

Macrophage Stimulating Protein ◽

Protein Receptor ◽

Ron Receptor ◽

Different Origins

The tyrosine kinase Ron, receptor for MSP (macrophage-stimulating protein), displays several serine residues of unknown functions. Using [32P]H3PO4 metabolic labelling, we found that Ron is serine-phosphorylated and dephosphorylated in vitro by PP1 (protein phosphatase 1). PP1 associates with Ron obtained from cells of different origins. The association is stimulated by MSP or serum and is prevented by wortmannin, an inhibitor of the Akt/PKB (protein serine/threonine kinase B) pathway. Akt/PKB phosphorylates Ron Ser-1394, thus providing a docking site for 14-3-3 (scaffold proteins binding to phosphoserine/phosphothreonine-containing sequences). In living cells, PP1 binds to the Ron mutant S1394A, but the association is no longer regulated by serum, MSP or wortmannin. The role of PP1 association with Ron is highlighted by (1) Ser-1394 dephosphorylation by PP1 in vitro and in living cells, (2) loss of 14-3-3 association with Ron after Ser-1394 dephosphorylation by PP1 in vitro and (3) an increase in 14-3-3 association after PP1 inactivation in living cells. These results suggest that PP1 can modulate the downstream Ron signalling generated by MSP via Akt/PKB and 14-3-3 binding. This is the first report on ligand-regulated association of PP1 with a growth factor receptor.

Download Full-text

The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction.

Molecular Biology of the Cell ◽

10.1091/mbc.e21-06-0307-t ◽

2021 ◽

Author(s):

Margaux R. Audett ◽

Erin L. Johnson ◽

Jessica M. McGory ◽

Dylan M. Barcelos ◽

Evelin Oroszne Szalai ◽

...

Keyword(s):

Protein Binding ◽

Protein Phosphatase ◽

Spindle Assembly Checkpoint ◽

Protein Phosphatase 1 ◽

Intrinsically Disordered ◽

Regulatory Circuit ◽

Cell Assays ◽

Kinetics Of

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of D. melanogaster KNL1 (Spc105) has never been shown to bind MTs nor to recruit PP1. Furthermore, the phospho-regulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell based-assays. A phospho-regulatory circuit, which utilizes Aurora B kinase (ABK), promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag, and deletion/chimera mutants are used to define the interplay of MT- and PP1-binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Download Full-text