Mapping the H+ (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation

Merkulova, Maria; Păunescu, Teodor G.; Azroyan, Anie; Marshansky, Vladimir; Breton, Sylvie; Brown, Dennis

doi:10.1038/srep14827

Cited by 113 publications

(149 citation statements)

References 61 publications

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“…SAINT assigns each protein a SAINT Probability Score ("P-Score"), on a scale of 0 to 1, with 1 being the top score. Based upon review of the literature, we chose 0.65 as a SAINT cut-off score for enrichment (68)(69)(70)(71)(72)(73). With the approach used here, all proteins have two P-Scores, one each for basal or insulin enrichment over the respective negative controls.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

Kruse

Krantz

Barker

et al. 2017

Molecular & Cellular Proteomics

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SummaryCLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane.To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes.Using two different commercially available antibodies for CLASP2 and an antibody for epitopetagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 co-immunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin.Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein.These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology. 5

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Section: Resultsmentioning

confidence: 99%

Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

Kruse

Krantz

Barker

et al. 2017

Molecular & Cellular Proteomics

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“…23 Under these conditions, there was no significant difference between untreated cells and those treated with 1 or 2 (Figure 4D). We had previously developed a more sensitive assay to study V-ATPase inhibition in cells that involves pre-treatment with bafilomycin and measuring vesicle re-acidification after it has been washed off.…”

mentioning

confidence: 86%

“…We had previously developed a more sensitive assay to study V-ATPase inhibition in cells that involves pre-treatment with bafilomycin and measuring vesicle re-acidification after it has been washed off. 23 Using this more sensitive assay, we determined the functional consequences of our electrophilic probes on V-ATPase-dependent vesicle acidification after exposing cells to bafilomycin (Figure S5). Immediately after bafilomycin washout, cells were treated with probe 1 at various concentrations, labeled with DND-99, followed by lysis and fluorescence measurement after 3 h. Probe 1 inhibited vesicle re-acidification with an apparent IC 50 value of 30 nM (Figure 4E), which aligns with the potency of its target occupancy measured by in-gel fluorescence (Figure 1B).…”

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confidence: 99%

Covalent Modulators of the Vacuolar ATPase

Chen¹,

Backus

Merkulova

et al. 2016

J. Am. Chem. Soc.

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The vacuolar H+ ATPase (V-ATPase) is a complex multi-subunit machine that regulates important cellular processes through controlling acidity of intracellular compartments in eukaryotes. Existing small-molecule modulators of V-ATPase either are restricted to targeting one membranous subunit of V-ATPase or have poorly understood mechanisms of action. Small molecules with novel and defined mechanisms of inhibition are thus needed to functionally characterize V-ATPase and to fully evaluate the therapeutic relevance of V-ATPase in human diseases. We have discovered electrophilic quinazolines that covalently modify a soluble catalytic subunit of V-ATPase with high potency and exquisite proteomic selectivity as revealed by fluorescence imaging and chemical proteomic activity-based profiling. The site of covalent modification was mapped to a cysteine residue located in a region of V-ATPase subunit A that is thought to regulate the dissociation of V-ATPase. We further demonstrate that a previously reported V-ATPase inhibitor, 3-bromopyruvate, also targets the same cysteine residue and that our electrophilic quinazolines modulate the function of V-ATPase in cells. With their well-defined mechanism of action and high proteomic specificity, the described quinazolines offer a powerful set of chemical probes to investigate the physiological and pathological roles of V-ATPase.

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“…2) [14]. The mechanism by which these important signaling molecules control V-ATPase assembly remains to be determined, although factors identified as interacting with the V-ATPase may be involved [37]. …”

Section: Regulation Of V-atpase Assembly In Dendritic Cellsmentioning

confidence: 99%

Regulation of V-ATPase assembly and function of V-ATPases in tumor cell invasiveness

McGuire

Cotter

Stransky

et al. 2016

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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V-ATPases are ATP-driven proton pumps that function within both intracellular compartments and the plasma membrane in a wide array of normal physiological and pathophysiological processes. V-ATPases are composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that transports protons. Regulated assembly of the V-ATPase represents an important mechanism of regulating V-ATPase activity in response to a number of environmental cues. Our laboratory has demonstrated that glucose-dependent assembly of the V-ATPase complex in yeast is controlled by the Ras/cAMP/PKA pathway. By contrast, increased assembly of the V-ATPase during dendritic cell maturation involves the PI-3 kinase and mTORC1 pathways. Recently, we have shown that amino acids regulate V-ATPase assembly in mammalian cells, possibly as a means to maintain adequate levels of amino acids upon nutrient starvation. V-ATPases have also been implicated in cancer cell survival and invasion. V-ATPases are targeted to different cellular membranes by isoforms of subunit a, with a3 targeting V-ATPases to the plasma membrane of osteoclasts. We have shown that highly invasive human breast cancer cell lines express higher levels of the a3 isoform than poorly invasive lines and that knockdown of a3 reduces both expression of V-ATPases at the plasma membrane and in vitro invasion of breast tumor cells. Moreover, overexpression of a3 in a non-invasive breast epithelial line increases both plasma membrane V-ATPases and in vitro invasion. Finally, specific ablation of plasma membrane V-ATPases in highly invasive human breast cancer cells using either an antibody or small molecule approach inhibits both in vitro invasion and migration. These results suggest that plasma membrane and a3-containing V-ATPases represent a novel and important target in the development of therapeutics to limit breast cancer metastasis.

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Mapping the H+ (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation

Cited by 113 publications

References 61 publications

Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein

Covalent Modulators of the Vacuolar ATPase

Regulation of V-ATPase assembly and function of V-ATPases in tumor cell invasiveness

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