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

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]

In Vivo and in Vitro Binding of Microcystin to Protein Phosphatase 1 and 2A

1995 ◽  
Vol 216 (1) ◽  
pp. 162-169 ◽  
Author(s):  
M. Runnegar ◽  
N. Berndt ◽  
S.M. Kong ◽  
E.Y.C. Lee ◽  
L.F. Zhang
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
In Vitro Binding ◽  
Vitro Binding

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

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

Protein Phosphatase 1 binds strongly to the retinoblastoma protein but not to p107 or p130 in vitro and in vivo

2002 ◽  
Vol 24 (5) ◽  
pp. 392-396 ◽  
Author(s):  
Joshua L. Dunaief ◽  
Ayala King ◽  
Noriko Esumi ◽  
Matthew Eagen ◽  
Tzvete Dentchev ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Retinoblastoma Protein ◽  
Protein Phosphatase 1

Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17

2003 ◽  
Vol 302 (2) ◽  
pp. 186-192 ◽  
Author(s):  
Thierry Dubois ◽  
Steven Howell ◽  
Eva Zemlickova ◽  
Michele Learmonth ◽  
Andy Cronshaw ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Phosphorylation Sites

scholarly journals Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

2007 ◽  
Vol 407 (2) ◽  
pp. 303-311 ◽  
Author(s):  
Benoit Devogelaere ◽  
Monique Beullens ◽  
Eva Sammels ◽  
Rita Derua ◽  
Etienne Waelkens ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Phosphorylation Sites ◽  
Docking Site ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Receptor Binding Protein

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1–104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.

scholarly journals Influence of substrates on in vitro dephosphorylation of glycogen phosphorylase a by protein phosphatase-1

1999 ◽  
Vol 341 (3) ◽  
pp. 545-554 ◽  
Author(s):  
Zhi-Xin WANG
Keyword(s):  
Protein Phosphatase ◽  
Glycogen Phosphorylase ◽  
Regulatory Mechanism ◽  
Protein Phosphatase 1 ◽  
Experimental Conditions ◽  
Enzyme Substrate ◽  
Novel Approach ◽  
Phosphorylation Reaction

The kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the dephosphorylation of phosphorylase a by protein phosphatase-1 (ppase-1). On the basis of the kinetic equation of the substrate reaction in the presence of ppase-1, all the inactivation rate constants for the free enzyme and the enzyme-substrate(s) complexes have been determined. Binding of the allosteric substrate, glucose 1-phosphate, to one subunit of phosphorylase a protects completely against ppase-1 action on either the same subunit or the adjacent subunit, whereas binding of the non-allosteric substrate, glycogen, to one subunit protects this subunit partially, but has no effect on the modification on the neighbouring subunit. Analysis of the data suggests that the allosteric behaviour of phosphorylase a can be interpreted in terms of a modified concerted model. The present method also provides a novel approach for studying dephosphorylation reactions. Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified, this new method would be particularly useful when the regulatory mechanism of the reversible phosphorylation reaction toward certain enzymes is being assessed.

scholarly journals Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons

1998 ◽  
Vol 330 (1) ◽  
pp. 211-216 ◽  
Author(s):  
Frédéric DESDOUITS ◽  
C. Julio SICILIANO ◽  
C. Angus NAIRN ◽  
Paul GREENGARD ◽  
Jean-Antoine GIRAULT
Keyword(s):  
Protein Phosphatase ◽  
Potent Inhibitor ◽  
Protein Phosphatases ◽  
Protein Phosphatase 1 ◽  
Protein Phosphatase 2C ◽  
Regulatory Cascade ◽  
Phosphatase 2A ◽  
Dependent Protein

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1.

scholarly journals Dephosphorylation of γH2A by Glc7/Protein Phosphatase 1 Promotes Recovery from Inhibition of DNA Replication

2009 ◽  
Vol 30 (1) ◽  
pp. 131-145 ◽  
Author(s):  
Marco Bazzi ◽  
Davide Mantiero ◽  
Camilla Trovesi ◽  
Giovanna Lucchini ◽  
Maria Pia Longhese
Keyword(s):  
Dna Replication ◽  
Protein Phosphatase ◽  
Replication Fork ◽  
Strand Break ◽  
Protein Phosphatase 1 ◽  
Synthesis Inhibitor ◽  
Replication Fork Stalling ◽  
Fork Stalling

ABSTRACT Replication fork stalling caused by deoxynucleotide depletion triggers Rad53 phosphorylation and subsequent checkpoint activation, which in turn play a crucial role in maintaining functional DNA replication forks. How cells regulate checkpoint deactivation after inhibition of DNA replication is poorly understood. Here, we show that the budding yeast protein phosphatase Glc7/protein phosphatase 1 (PP1) promotes disappearance of phosphorylated Rad53 and recovery from replication fork stalling caused by the deoxynucleoside triphosphate (dNTP) synthesis inhibitor hydroxyurea (HU). Glc7 is also required for recovery from a double-strand break-induced checkpoint, while it is dispensable for checkpoint inactivation during methylmethane sulfonate exposure, which instead requires the protein phosphatases Pph3, Ptc2, and Ptc3. Furthermore, Glc7 counteracts in vivo histone H2A phosphorylation on serine 129 (γH2A) and dephosphorylates γH2A in vitro. Finally, the replication recovery defects of HU-treated glc7 mutants are partially rescued by Rad53 inactivation or lack of γH2A formation, and the latter also counteracts hyperphosphorylated Rad53 accumulation. We therefore propose that Glc7 activity promotes recovery from replication fork stalling caused by dNTP depletion and that γH2A dephosphorylation is a critical Glc7 function in this process.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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