Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

Recombinant FlagHis[subscript 6] tagged Human P2X1 receptors expressed in HEK293 cells were purified, digested with trypsin and analysed by mass spectroscopy (96% coverage following de-glycosylation and reduction). The receptor was basally phosphorylated at residues S387, S388 and T389 in the carboxyl terminus, a triple alanine mutant of these residues had a modest ~ 25% increase in current amplitude and recovery from desensitization. Chemical modification showed that intracellular lysine residues close to the transmembrane domains and the membrane stabilization motif are accessible to the aqueous environment. The membrane-impermeant cross-linking reagent 3,3′-Dithiobis (sulfosuccinimidylpropionate) (DTSSP) reduced agonist binding and P2X1 receptor currents by > 90%, and modified lysine residues were identified by mass spectroscopy. Mutation to remove reactive lysine residues around the ATP-binding pocket had no effect on inhibtion of agonist evoked currents following DTSSP. However, agonist evoked currents were ~ 10-fold higher than for wild type following DTSSP treatment for mutants K199R, K221R and K199R-K221R. These mutations remove reactive residues distant from the agonist binding pocket that are close enough to cross-link adjacent subunits. These results suggest that conformational change in the P2X1 receptor is required for co-ordination of ATP action

Transcriptional and Post-Translational Targeting of Myocyte Stress Protein 1 (MS1) by the JNK Pathway in Cardiac Myocytes.

Myocyte Stress Protein 1 (MS1) is a muscle-specific, stress-responsive, regulator of gene expression. It was originally identified in embryonic mouse heart which showed increased expression in a rat model of left ventricular hypertrophy. To determine if MS1 was responsive to other stresses relevant to cardiac myocyte function, we tested if it could be induced by the metabolic stresses associated with ischaemia/reperfusion injury in cardiac myocytes. We found that metabolic stress increased MS1 expression, both at the mRNA and protein level, concurrent with activation of the c-Jun N-terminal Kinase (JNK) signalling pathway. MS1 induction by metabolic stress was blocked by both the transcription inhibitor actinomycin D and a JNK inhibitor, suggesting that activation of the JNK pathway during metabolic stress in cardiac myocytes leads to transcriptional induction of MS1. MS1 was also found to be an efficient JNK substrate in vitro, with a major JNK phosphorylation site identified at Thr-62. In addition, MS1 was found to co-precipitate with JNK, and inspection of the amino acid sequence upstream of the phosphorylation site, at Thr-62, revealed a putative Mitogen-Activated Protein Kinase (MAPK) binding site. Taken together, these data identify MS1 as a likely transcriptional and post-translational target for the JNK pathway in cardiac myocytes subjected to metabolic stress

PfCK2 phosphorylates MCM2 on Ser13 and Tyr16 <i>in vitro</i>.

<p><b>A</b>: In vitro kinase assay using a GST fusion protein containing a N-terminal portion of MCM2 (GST-MCM2) or the same fusion protein but where residue Y16 is mutated to an phenylalanine (Y16F) or where residue S13 is mutated to an alanine (S13A) or where both S13 and Y16 are mutated to an alanine and phenylalanine respectively (S13A/Y16F). Top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: LC-MS/MS trace of the fusion protein GST-PfMCM2 containing the S13 to alanine mutation following phosphorylation with PfCK2 indicating the phosphorylation of residue Y16. Also shown is the fragmentation table (detected b-ions and y-ions are represented respectively in bold red and bold blue). <b>C</b>: N-terminal sequence of PfMCM2 protein showing the phospho-peptide identified in the LC-MS/MS analysis that contains the tyrosine phosphorylated residue (in red).</p

PfCK2 auto-phosphorylates <i>in vitro</i> on threonine 63.

<p><b>A</b>: <i>In vitro</i> kinase assay for GST-PfCK2 autophosphorylation, top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: LC-MS/MS trace identifying phosphorylation of PfCK2 at T63; right: Also shown is the hypothetical fragmentation table where the b-ions and y-ions detected in the LC-MS/MS spectra are shown in red and bold, respectively. <b>C</b>: Sequence of PfCK2 showing the phosphopeptide identified in the LC-MS/MS analysis (underlined) and the threonine 63 phosphorylation site (in red).</p

Autophosphorylation of PfCK2 regulates kinase activity.

<p>The activity of PfCK2α and a mutant PfCK2α where threonine 63 was mutated to alanine (T63A) was tested in <i>in vitro</i> kinase assays using α-casein as a substrate. <b>A</b>: Example of the <i>in vitro</i> kinase assay with PfCK2α and the T63A mutant. Top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: kinase activity quantification. Date represents the mean ± S.E.M (n = 3) <b>C</b>: LC-MS/MS trace of PfCK2 identifying T63 phosphorylation from a shizont stage lysate of <i>P. falciparum</i>. Indicated are the b-ions and b-ions (−98daltons) that were identified in the LC-MS/MS spectra. Also shown is the hypothetical fragmentation table where the ions that were identified in the LC-MS/MS spectra are shown in red.</p

Structural analysis of PfCK2α inhibition by quinalizarin.

<p><b>A</b>: <i>In vitro</i> inhibition assay showing the affect of various concentrations of quinalizarin on the activity of human protein kinase CK2α (red) and PfCK2α (blue). Date represents the mean ± S.E.M (n = 3) <b>B</b>: Superimposition of the calculate <i>in silico</i> homology model for PfCK2α (purple) with the <i>Zea mays</i> protein kinase CK2α crystal structure (green, PDB code: 3FL5, resolution 2.30 Å); <b>B</b>: Superimposition of the molecular docking of the PfCK2 homology model with quinalizarin (purple) and the co-crystal structure of <i>Z. mays</i> protein kinase CK2α and quinalizarin (green); non-conserved residues are indicated in bold.</p

A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5' end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs

An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture

Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion