scholarly journals Characterization of Rictor Phosphorylation Sites Reveals Direct Regulation of mTOR Complex 2 by S6K1

2009 ◽  
Vol 29 (21) ◽  
pp. 5657-5670 ◽  
Author(s):  
Christian C. Dibble ◽  
John M. Asara ◽  
Brendan D. Manning
Keyword(s):  
Control Cell ◽  
Mammalian Target Of Rapamycin ◽  
Phosphorylation Site ◽  
Phosphorylation Sites ◽  
Mediated Effects ◽  
Phosphorylation Event ◽  
Liquid Chromatography Tandem Mass ◽  
Precise Molecular Mechanism

ABSTRACT The mammalian target of rapamycin (mTOR) functions within two distinct complexes (mTORC1 and mTORC2) to control cell growth, proliferation, survival, and metabolism. While there has been great progress in our understanding of mTORC1 regulation, the signaling mechanisms that regulate mTORC2 have not been defined. In this study, we use liquid chromatography-tandem mass spectrometry analyses to identify 21 phosphorylation sites on the core mTORC2 component Rictor. We find that one site, T1135, undergoes growth factor-responsive phosphorylation that is acutely sensitive to rapamycin and is phosphorylated downstream of mTORC1. We find that Rictor-T1135 is directly phosphorylated by the mTORC1-dependent kinase S6K1. Although this phosphorylation event does not affect mTORC2 integrity or in vitro kinase activity, expression of a phosphorylation site mutant of Rictor (T1135A) in either wild-type or Rictor null cells causes an increase in the mTORC2-dependent phosphorylation of Akt on S473. However, Rictor-T1135 phosphorylation does not appear to regulate mTORC2-mediated effects on SGK1 or PKCα. While the precise molecular mechanism affecting Akt is unknown, phosphorylation of T1135 stimulates binding of Rictor to 14-3-3 proteins. We provide evidence that Rictor-T1135 phosphorylation acts in parallel with other mTORC1-dependent feedback mechanisms, such as those affecting IRS-1 signaling to PI3K, to regulate the response of Akt to insulin.

scholarly journals Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

1991 ◽  
Vol 279 (3) ◽  
pp. 727-732 ◽  
Author(s):  
G B Sala-Newby ◽  
A K Campbell
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Live Cells ◽  
Phosphorylation Sites ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

scholarly journals Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5

2009 ◽  
Vol 83 (18) ◽  
pp. 9195-9205 ◽  
Author(s):  
Dipankar Bhattacharya ◽  
Mayuri ◽  
S. M. Best ◽  
R. Perera ◽  
R. J. Kuhn ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Yellow Fever Virus ◽  
Nonstructural Protein ◽  
Protein Kinase G ◽  
Phosphorylation Sites ◽  
Histidine Residue ◽  
Phosphorylation Event ◽  
Langat Virus ◽  
And Function

ABSTRACT Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.

scholarly journals Differential phosphorylation signals control endocytosis of GPR15

2017 ◽  
Vol 28 (17) ◽  
pp. 2267-2281 ◽  
Author(s):  
Yukari Okamoto ◽  
Sojin Shikano
Keyword(s):  
Surface Density ◽  
Functional Role ◽  
Control Cell ◽  
Phosphorylation Site ◽  
Hek293 Cells ◽  
C Terminus ◽  
Differential Phosphorylation ◽  
G Protein Coupled

GPR15 is an orphan G protein–coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the β-arrestin– and GPCR kinase–dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis.

scholarly journals The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2053
Author(s):  
Dalma Ménesi ◽  
Éva Klement ◽  
Györgyi Ferenc ◽  
Attila Fehér
Keyword(s):  
Protein Kinases ◽  
Phosphorylation Site ◽  
Interaction Strength ◽  
Yeast Cells ◽  
Phosphorylation Sites ◽  
Mutant Proteins ◽  
In Vivo Test ◽  
Calcium Dependent

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.

scholarly journals Minireview: The Busy Road to Pheochromocytomas and Paragangliomas Has a New Member, TMEM127

Endocrinology ◽  
2011 ◽  
Vol 152 (6) ◽  
pp. 2133-2140 ◽  
Author(s):  
Shoulei Jiang ◽  
Patricia L. M. Dahia
Keyword(s):  
Transmembrane Protein ◽  
Functional Characterization ◽  
Susceptibility Gene ◽  
Mammalian Target Of Rapamycin ◽  
Target Of Rapamycin ◽  
Molecular Defect ◽  
Endocrine Tumors ◽  
Functional Classes

Characterization of the entire spectrum of cancer-associated genetic disruptions is an overarching goal of contemporary and future oncology and can inform on patient diagnosis, treatment, and surveillance. Hereditary endocrine tumors, by having the potential to reveal the cancer's primary molecular defect, have been especially informative in this realm. Within this group, pheochromocytomas and paragangliomas, neural crest-derived, catecholamine-secreting tumors have come to represent true conduits for gene discovery. About one-third of pheochromocytomas and paragangliomas are now known to result from germline mutations in one of at least eight genes that belong to a variety of functional classes. Greater understanding of the molecular signals transduced by these genes and their respective mutants has advanced our understanding of kinase signaling pathways, hypoxia regulation, and the link between metabolic disruptions and cell growth. A new susceptibility gene without homology to other functional classes has been recently identified and encodes for a three-spanner transmembrane protein, transmembrane protein 127 (TMEM127). Initial insights from in vitro and patient data suggest that this candidate tumor suppressor is linked to the endosomal system and the mechanistic target of rapamycin [formerly mammalian target of rapamycin (mTOR)] pathway, and that mutation carriers often have clinical features that are typically associated with sporadic forms of pheochromocytoma. Functional characterization of transmembrane protein 127 (TMEM127) and discovery of additional pheochromocytoma/paraganglioma susceptibility genes is likely to shed light on our understanding of these tumors and extend these insights to other cancers.

scholarly journals An Artificial Intelligence Characterised Functional Ingredient, Derived from Rice, Inhibits TNF-α and Significantly Improves Physical Strength in an Inflammaging Population

Foods ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1147
Author(s):  
Kathy Kennedy ◽  
Brian Keogh ◽  
Cyril Lopez ◽  
Alessandro Adelfio ◽  
Brendan Molloy ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Functional Ingredient ◽  
Physical Strength ◽  
Active Peptides ◽  
Tnf Α ◽  
Liquid Chromatography Tandem Mass ◽  
Natural Peptide ◽  
Objectively Measured

Food-derived bioactive peptides offer great potential for the treatment and maintenance of various health conditions, including chronic inflammation. Using in vitro testing in human macrophages, a rice derived functional ingredient natural peptide network (NPN) significantly reduced Tumour Necrosis Factor (TNF)-α secretion in response to lipopolysaccharides (LPS). Using artificial intelligence (AI) to characterize rice NPNs lead to the identification of seven potentially active peptides, the presence of which was confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Characterization of this network revealed the constituent peptides displayed anti-inflammatory properties as predicted in vitro. The rice NPN was then tested in an elderly “inflammaging” population with a view to subjectively assess symptoms of digestive discomfort through a questionnaire. While the primary subjective endpoint was not achieved, analysis of objectively measured physiological and physical secondary readouts showed clear significant benefits on the ability to carry out physical challenges such as a chair stand test that correlated with a decrease in blood circulating TNF-α. Importantly, the changes observed were without additional exercise or specific dietary alterations. Further health benefits were reported such as significant improvement in glucose control, a decrease in serum LDL concentration, and an increase in HDL concentration; however, this was compliance dependent. Here we provide in vitro and human efficacy data for a safe immunomodulatory functional ingredient characterized by AI.

scholarly journals Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

1996 ◽  
Vol 7 (10) ◽  
pp. 1573-1586 ◽  
Author(s):  
K L Gould ◽  
A Feoktistova
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Phosphorylation Site ◽  
Loss Of Function ◽  
Contractile Ring ◽  
Mutant Proteins ◽  
Regulatory Phosphorylation ◽  
Binding Loop

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.

scholarly journals Positive/negative ion-switching-based LC–MS/MS method for quantification of cytosine derivatives produced by the TET-family 5-methylcytosine dioxygenases

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Aninda Sundar Dey ◽  
Navid J Ayon ◽  
Chayan Bhattacharya ◽  
William G Gutheil ◽  
Mridul Mukherji
Keyword(s):  
Mass Spectrometry ◽  
Cytosine Methylation ◽  
Negative Ion ◽  
Tandem Mass ◽  
Active Demethylation ◽  
Chromatography Tandem Mass Spectrometry ◽  
In Vitro Characterization ◽  
Liquid Chromatography Tandem Mass

Abstract Cytosine methylation at carbon-5 (5mC) in DNA plays crucial roles in epigenetic transcriptional regulation during metazoan development. The iron (II), 2-oxoglutarate-dependent Ten-Eleven Translocation (TET)-family dioxygenases initiate active demethylation of 5mC. TET2 oxidizes 5mC in nucleic acids into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine by iterative oxidation. Mutations in the TET2 gene are frequently detected in myeloid malignancies. Despite the established and emerging roles of TET oxygenases in health and diseases, in vitro characterization of these enzymes and their mutants is still in rudimentary stages. Here, we describe an improved positive/negative ion-switching-based liquid chromatography-tandem mass spectrometry (LC–MS/MS) method that can separate and quantify modified cytosine bases produced by TET-family 5-methylcytosine dioxygenases. This method will help in further elucidate the function of epigenetically important cytosine modifications. To the best of our knowledge, this is the first study reporting ion-switching-based LC–MS/MS method to analyse cytosine variants produced in TET catalysed reactions.

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