scholarly journals A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition

2017 ◽  
Author(s):  
Hannah R. Belsham ◽  
Claire T. Friel
Keyword(s):  
Molecular Mechanism ◽  
Residence Time ◽  
Phosphorylation Site ◽  
Motor Domain ◽  
Wild Type ◽  
Metaphase Arrest ◽  
Specific Stimulation ◽  
The Impact ◽  
Stimulation Of

AbstractThe microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAK’s motor domain. To understand the impact of phosphorylation by Cdk1 microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.

scholarly journals A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e4034 ◽  
Author(s):  
Hannah R. Belsham ◽  
Claire T. Friel
Keyword(s):  
Molecular Mechanism ◽  
Residence Time ◽  
Phosphorylation Site ◽  
Motor Domain ◽  
Wild Type ◽  
Metaphase Arrest ◽  
Specific Stimulation ◽  
The Impact ◽  
Stimulation Of

The microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAK’s motor domain. To understand the impact of phosphorylation by Cdk1 on microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.

scholarly journals Synthetic-evolution reveals that phosphoregulation of the mitotic kinesin-5 Cin8 is constrained

2018 ◽  
Author(s):  
Alina Goldstein ◽  
Darya Goldman ◽  
Ervin Valk ◽  
Mart Loog ◽  
Liam J. Holt ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phosphorylation Site ◽  
Motor Domain ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Site Specific ◽  
Precise Positioning ◽  
Optimal Regulation

AbstractCdk1 has been found to phosphorylate the majority of its substrates in disordered regions. These phosphorylation sites do not appear to require precise positioning for their function. The mitotic kinesin-5 Cin8 was shown to be phosphoregulated at three Cdk1 sites in disordered loops within its catalytic motor domain. Here, we examined the flexibility of Cin8 phosphoregulation by analyzing the phenotypes of synthetic Cdk1-sites that were systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites that we created, eight were non-functional; 19 were neutral, similar to the phosphodeficient variant; and two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one site resulted in novel phosphoregulation, and only one site, immediately adjacent to a native Cdk1 site, produced phosphoregulation similar to wild-type. This study shows that, while the gain of a single phosphorylation site can confer regulation and modulate the dynamics of the spindle, to achieve optimal regulation of a mitotic kinesin-5 motor protein, phosphoregulation has to be site-specific and precise.

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase

1996 ◽  
Vol 270 (1) ◽  
pp. C131-C137 ◽  
Author(s):  
P. Beguin ◽  
A. Beggah ◽  
S. Cotecchia ◽  
K. Geering
Keyword(s):  
Recombinant Dna ◽  
Phosphorylation Site ◽  
Alpha Subunit ◽  
Receptor Interaction ◽  
Wild Type ◽  
Dna Encoding ◽  
Muscarinic Cholinergic ◽  
Beta 2 ◽  
G Protein Coupled ◽  
Stimulation Of

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.

Overexpression of ADP-glucose pyrophosphorylase in both leaf and seed tissue synergistically increase biomass and seed number in rice (Oryza sativa ssp. japonica)

2016 ◽  
Vol 43 (12) ◽  
pp. 1194 ◽  
Author(s):  
Alanna J. Oiestad ◽  
John M. Martin ◽  
Michael J. Giroux
Keyword(s):  
Plant Growth ◽  
Carbon Metabolism ◽  
Plant Biomass ◽  
Flag Leaf ◽  
Starch Biosynthesis ◽  
Wild Type ◽  
Whole Plant ◽  
Panicle Number ◽  
The Impact ◽  
Stimulation Of

Increased expression of leaf or seed ADPglucose pyrophosphorylase activity (AGPase) has been shown to increase plant growth. However, no study has directly compared AGPase overexpression in leaves and/or seeds. In the present study, transgenic rice overexpressing AGPase in leaves or in seeds were crossed, resulting in four F2:3 homozygous genotypes with AGPase overexpression in leaves, seeds, both leaves and seeds, or neither tissue. The impact of AGPase overexpression in these genotypic groups was examined at the metabolic, transcriptomic, and plant growth levels. Leaf-specific AGPase overexpression increased flag leaf starch up to five times that of the wild type (WT) whereas overexpression of AGPase in both leaves and seeds conferred the greatest productivity advantages. Relative to the WT, AGPase overexpression in both leaves and seeds increased plant biomass and panicle number by 61% and 51%, respectively while leaf-specific AGPase overexpression alone only increased plant biomass and panicle number by 24 and 32% respectively. Extraction and analysis of RNA and leaf-specific metabolites demonstrated that carbon metabolism was broadly increased by AGPase overexpression in seeds and leaves. These findings indicate that stimulation of whole-plant growth and productivity can be best achieved by upregulation of starch biosynthesis in both leaves and seeds.

Subtle Impact of Akt1 and Akt3 on Exploratory Behavior in Gene Targeted Mice

2015 ◽  
Vol 223 (3) ◽  
pp. 173-180 ◽  
Author(s):  
Christina Leibrock ◽  
Michael Hierlmeier ◽  
Undine E. Lang ◽  
Florian Lang
Keyword(s):  
Forced Swimming Test ◽  
Open Field Test ◽  
Wild Type ◽  
Average Speed ◽  
Closed Area ◽  
Light Area ◽  
Swimming Test ◽  
The Impact ◽  
Center Area ◽  
Dark Transition

Abstract. The present study explored the impact of Akt1 and Akt3 on behavior. Akt1 (akt1-/-) and Akt3 (akt3-/-) knockout mice were compared to wild type (wt) mice. The akt1-/- mice, akt3-/- mice, and wt mice were similar in most parameters of the open-field test. However, the distance traveled in the center area was slightly but significantly less in akt3-/- mice than in wt mice. In the light/dark transition test akt1-/- mice had significantly lower values than wt mice and akt3-/- mice for distance traveled, number of rearings, rearing time in the light area, as well as time spent and distance traveled in the entrance area. They were significantly different from akt3-/- mice in the distance traveled, visits, number of rearings, rearing time in the light area, as well as time spent, distance traveled, number of rearings, and rearing time in the entrance area. In the O-maze the time spent, and the visits to open arms, as well as the number of protected and unprotected headdips were significantly less in akt1-/- mice than in wt mice, whereas the time spent in closed arms was significantly more in akt1-/- mice than in wt mice. Protected and unprotected headdips were significantly less in akt3-/- mice than in wt mice. In closed area, akt3-/- mice traveled a significantly larger distance at larger average speed than akt1-/- mice. No differences were observed between akt1-/- mice, akt3-/- mice and wt-type mice in the time of floating during the forced swimming test. In conclusion, akt1-/- mice and less so akt3-/ mice display subtle changes in behavior.

Influence of DAFS-25+polizon composition on blood and functional state of liver of steers at fattening

2020 ◽  
pp. 15-18
Author(s):  
Inna R. Kilmetova ◽  
◽  
Igor A. Rodin ◽  
Nazira I. Khayrullina ◽  
Nikolay G. Fenchenko ◽  
...  
Keyword(s):  
Functional State ◽  
White Blood Cells ◽  
Live Weight ◽  
The Body ◽  
Farm Animals ◽  
Control Group ◽  
Standard Diet ◽  
The Impact ◽  
Experimental Group ◽  
Stimulation Of

Summary. The disbalanced feeding and the uneven distribution of micro- and macroelements in the environment leads to a trace element, in particular hypomelanosis. To accelerate the growth and preservation of young farm animals include in the diet of various biological additives and drugs, which include selenium. For stimulation of weight gain in the livestock industry, as well as for the prevention and treatment of pathological processes in addition to micro - and macrouse amino acids, primarily methionine. The aim of this work was to study the influence of composition of DAFS-25+Polizon on morpho-biochemical parameters of blood and functional state of the liver in fattening bulls of black-motley breed in the conditions of the Republic of Bashkortostan. Experiments using were conducted on bull-calves of black-motley breed of the properties in the properties age from 6 to 15 months. The first experimental group during the experiment was additionally given the composition of DAFS-25+Polizon at a dose of 2 mg/kg, the animals of the control group received a standard diet. To assess the impact of the composition DAFS-25+Polizon on metabolism cattle studied morphological and biochemical indicators of blood and conducted histological examination of the liver. It is established that the use of the composition of DAFS-25+Polizon at a dose of 2 mg/kg increases the number of erythrocytes and hemoglobin in the experimental group and reduces the amount of white blood cells. The serum content of total protein, phosphorus and calcium increases in the group of experimental animals. Microscopic examination of the liver revealed no changes in the structure of the organ and hepatocytes in the experimental group, whereas in the control group hemodynamic disorders and dystrophic changes in liver cells were observed. Thus, the use of the composition DAFS-25+Polizon at a dose of 2 mg/kg of live weight in fattening bulls black-and-white breed contributes to the increase of redox processes in the body, stimulation of metabolism, prevent the development of liver disorders of cellular mechanisms of metabolism, optimizes the structure of the liver, which generally provides higher productivity.

scholarly journals Titanium Dioxide Presents a Different Profile in Dextran Sodium Sulphate-Induced Experimental Colitis in Mice Lacking the IBD Risk Gene Ptpn2 in Myeloid Cells

2021 ◽  
Vol 22 (2) ◽  
pp. 772
Author(s):  
Javier Conde ◽  
Marlene Schwarzfischer ◽  
Egle Katkeviciute ◽  
Janine Häfliger ◽  
Anna Niechcial ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Titanium Dioxide ◽  
Genetic Risk ◽  
Intestinal Inflammation ◽  
Sodium Sulphate ◽  
Food Additive ◽  
Wild Type ◽  
Dextran Sodium Sulphate ◽  
Risk Gene ◽  
The Impact

Environmental and genetic factors have been demonstrated to contribute to the development of inflammatory bowel disease (IBD). Recent studies suggested that the food additive; titanium dioxide (TiO2) might play a causative role in the disease. Therefore, in the present study we aimed to explore the interaction between the food additive TiO2 and the well-characterized IBD risk gene protein tyrosine phosphatase non-receptor type 2 (Ptpn2) and their role in the development of intestinal inflammation. Dextran sodium sulphate (DSS)-induced acute colitis was performed in mice lacking the expression of Ptpn2 in myeloid cells (Ptpn2LysMCre) or their wild type littermates (Ptpn2fl/fl) and exposed to the microparticle TiO2. The impact of Ptpn2 on TiO2 signalling pathways and TiO2-induced IL-1β and IL-10 levels were studied using bone marrow-derived macrophages (BMDMs). Ptpn2LysMCre exposed to TiO2 exhibited more severe intestinal inflammation than their wild type counterparts. This effect was likely due to the impact of TiO2 on the differentiation of intestinal macrophages, suppressing the number of anti-inflammatory macrophages in Ptpn2 deficient mice. Moreover, we also found that TiO2 was able to induce the secretion of IL-1β via mitogen-activated proteins kinases (MAPKs) and to repress the expression of IL-10 in bone marrow-derived macrophages via MAPK-independent pathways. This is the first evidence of the cooperation between the genetic risk factor Ptpn2 and the environmental factor TiO2 in the regulation of intestinal inflammation. The results presented here suggest that the ingestion of certain industrial compounds should be taken into account, especially in individuals with increased genetic risk

scholarly journals The Arabidopsis Root Tip (Phospho)Proteomes at Growth-Promoting versus Growth-Repressing Conditions Reveal Novel Root Growth Regulators

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1665
Author(s):  
Natalia Nikonorova ◽  
Evan Murphy ◽  
Cassio Flavio Fonseca de Lima ◽  
Shanshuo Zhu ◽  
Brigitte van de Cotte ◽  
...  
Keyword(s):  
Cell Wall ◽  
Root Growth ◽  
Growth Regulators ◽  
Growth Regulation ◽  
Phosphorylation Site ◽  
Primary Root ◽  
Root Tip ◽  
Arabidopsis Root ◽  
Growth Promoting ◽  
The Impact

Auxin plays a dual role in growth regulation and, depending on the tissue and concentration of the hormone, it can either promote or inhibit division and expansion processes in plants. Recent studies have revealed that, beyond transcriptional reprogramming, alternative auxin-controlled mechanisms regulate root growth. Here, we explored the impact of different concentrations of the synthetic auxin NAA that establish growth-promoting and -repressing conditions on the root tip proteome and phosphoproteome, generating a unique resource. From the phosphoproteome data, we pinpointed (novel) growth regulators, such as the RALF34-THE1 module. Our results, together with previously published studies, suggest that auxin, H+-ATPases, cell wall modifications and cell wall sensing receptor-like kinases are tightly embedded in a pathway regulating cell elongation. Furthermore, our study assigned a novel role to MKK2 as a regulator of primary root growth and a (potential) regulator of auxin biosynthesis and signalling, and suggests the importance of the MKK2 Thr31 phosphorylation site for growth regulation in the Arabidopsis root tip.

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