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A mass spectrometry-based predictive strategy reveals ADAP1 is phosphorylated at Tyrosine 364.

ABSTRACT: Abstract Rationale The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry-based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition. Methods ADAP1 was immunoprecipitated from extracts of HEK293 cells stably-transfected with ADAP1 cDNA. Immunoprecipitated ADAP1 was digested with proteolytic enzymes and analyzed by LC-MS in MS1 mode by high-resolution quadrupole time-of-flight mass spectrometry (QTOF-MS). Peptide molecular features were extracted using an untargeted data mining algorithm. Extracted peptide neutral masses were matched against the ADAP1 amino acid sequence with phosphorylation included as a predicted modification. Peptides with predicted phosphorylation sites were analyzed by targeted LC-MS2. Acquired MS2 spectra were then analyzed using database search engines to confirm phosphorylation. Spectra of phosphorylated peptides were validated by manual interpretation. Further confirmation was performed by manipulating phospho-peptide abundance using calf intestinal phosphatase (CIP) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Results Of five predicted phosphopeptides, one, comprised of the sequence AVDRPMLPQEYAVEAHFK, was confirmed to be phosphorylated on a Tyrosine at position 364. Pre-treatment of cells with PMA prior to immunoprecipitation increased the ratio of phosphorylated to unphosphorylated peptide as determined by area counts of extracted ion chromatograms (EIC). Addition of CIP to immunoprecipitation reactions eliminated the phosphorylated form. Conclusions A novel phosphorylation site was identified at Tyrosine 364. Phosphorylation at this site is increased by treatment with PMA. PMA promotes membrane translocation and activation of protein kinase C (PKC), indicating that Tyrosine 364 is phosphorylated by a PKC-dependent mechanism.

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Early Embryonic Cell, Hela Cell

SUBMITTER: Rick Reisdorph

LAB HEAD: Richard M. Reisdorph

PROVIDER: PXD009252 | Pride | 2018-04-26

REPOSITORIES: Pride

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A mass spectrometry based predictive strategy reveals ADAP1 is phosphorylated at tyrosine 364.

Reisdorph Richard R Littrell-Miller BobbiJo B Powell Roger R Reisdorph Nichole N

Rapid communications in mass spectrometry : RCM 20180801 15

<h4>Rationale</h4>The goal of this work was to identify phosphorylation sites within the amino acid sequence of human ADAP1. Using traditional mass spectrometry based techniques we were unable to produce interpretable spectra demonstrating modification by phosphorylation. This prompted us to employ a strategy in which phosphorylated peptides were first predicted using peptide mapping followed by targeted MS/MS acquisition.<h4>Methods</h4>ADAP1 was immunoprecipitated from extracts of HEK293 cells ...[more]

PMID: 29659066

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Project description:Human histomonocytic U937 cells exhibit macrophage-like properties after phorbol ester stimulation. Accumulating evidence demonstrated that remarkable number of transcripts changed in their amount at total RNA levels. However, post-transcriptional regulation has not been fully elucidated in this model. Thus, we compared expression profiles between polysomal and cytoplasmic RNAs before and after phorbol 12-myristate 13-acetate stimulation (48h, 32nM). Table 1: Detailed description of the expression data of human histomonocytic cell line U937 cells. Data of total cytoplasmic and polysomal fractions before and after PMA stimulation is shown. This table contains all spot data except: 1) saturated spots, 2) undetectable spots, 3) stained spots, and 4) spots only detected in less than one samples. Column A: Gene symbol (“I_xxxxxx” is corresponding to EST clones) Column B: Genbank accession no. Column C: Description Column D: Average value of expression levels in total cytoplasmic fraction in the absence of PMA Column E: Average value of expression levels in total cytoplasmic fraction in the presence of PMA (32nM, 48h) Column F: Average value of expression levels in polysomal fraction in the absence of PMA Column G: Average value of expression levels in polysomal fraction in the presence of PMA Column H: Expression ratio of polysome, PMA (-) to cytoplasm, PMA(-) Column I: Expression ratio of polysome, PMA (+) to cytoplasm, PMA(+) Column J: Expression ratio of cytoplasm, PMA (+) to cytoplasm, PMA(-) Column K: Expression ratio of polysome, PMA (+) to polysome, PMA(-) Column L: Different expression between polysome, PMA (-) and cytoplasm, PMA(-): different=1 Column M: Different expression between polysome, PMA (+) and cytoplasm, PMA(+): different=1 Column N: Different expression between cytoplasm, PMA (+) and cytoplasm, PMA(-): different=1 Column O: Different expression between polysome, PMA (+) and polysome, PMA(-): different=1 Table 2: Candidates post-transcriptionally regulated by PMA. We extracted 105 transcripts whose expression levels were altered only in either fraction accompanied by changes in polysome/cytoplasm expression ratio. Column A: Gene symbol (“I_xxxxxx” is corresponding to EST clones) Column B: Genbank accession no. Column C: Description Column D: Average value of expression levels in total cytoplasmic fraction in the absence of PMA Column E Average value of expression levels in total cytoplasmic fraction in the presence of PMA Column F: Average value of expression levels in polysomal fraction in the absence of PMA Column G: Average value of expression levels in polysomal fraction in the presence of PMA Column H: Expression ratio of polysome, PMA (-) to cytoplasm, PMA(-) Column I: Expression ratio of polysome, PMA (+) to cytoplasm, PMA(+) Column J: Expression ratio of cytoplasm, PMA (+) to cytoplasm, PMA(-) Column K: Expression ratio of polysome, PMA (+) to polysome, PMA(-) Keywords = phorbol ester Keywords = macrophage Keywords = polysome Keywords = post-transcriptional regulation Keywords: parallel sample

Project description:Background: Our purpose was to obtain non-existing genome-wide expression data for the rabbit species on the responses of peripheral blood mononuclear cells (PBMCs) after in vitro stimulation by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) and ionomycin. This transcriptomic study was carried out using microarrays enriched with immunity-related genes, and annotated with the most recent data available for the rabbit genome. Results: The LPS affected 15 to 20 less genes than PMA/Ionomycin after 4 hours (T4) or 24 hours (T24), of in vitro stimulation, by comparison with mock-stimulated PBMCs. LPS induced an inflammatory response as shown by a significant up-regulation of IL12A and CXCL11 at T4, followed by an increased transcription of IL6, IL1B, IL1A, IL36, IL37, TNF, CCL4 and SAA1 at T24. Surprisingly, we could not find an up-regulation of IL8 either at T4 or at T24. PMA/ionomycin induced a very early expression of Th1, Th2, iTreg, and Th17 responses by PBMCs at T4. The Th1 response was increased at T24 as shown by the increase of the transcription of IFNG and by contrast to other cytokines that significantly decreased from T4 to 24 (IL2, IL4, IL10, IL13, IL17A, CD69). The granulocyte-macrophage colony-stimulating factor (CSF2) was by far the most over-expressed gene at both T4 and T24 by comparison to mock-stimulated cells, confirming a major impact of PMA/ionomycin on cell growth and proliferation. A significant down-regulation of IL16 was observed at T4 and T24, in agreement with a role of IL16 in in PBMC apoptosis. Conclusion: We report new data on the responses of PBMCs to LPS and PMA/ionomycin for the rabbit species, thus enlarging the set of mammalian species for which such reports exist. The availability of the rabbit genome assembly together with highly performing genomic tools should pave the way for more intense genomic studies for this species known to be a very relevant animal model in immunology and physiology. To characterize gene expression changes in peripheral blood mononuclear cells (PBMCs), we collected blood from 4 adult rabbits. PBMCs were isolated and stimulated with LPS or a mixture of PMA/Ionomycin, versus a control group (C). Cells were harvested either 4h or 24h after activation.

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A mass spectrometry-based predictive strategy reveals ADAP1 is phosphorylated at Tyrosine 364.

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A mass spectrometry based predictive strategy reveals ADAP1 is phosphorylated at tyrosine 364.

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