Quantitative phosphoproteomic analysis of the PI3K-regulated signaling network

Gnad, Florian; Wallin, Jeffrey J.; Edgar, Kyle A.; Doll, Sophia; Arnott, David; Robillard, Liliane; Kirkpatrick, Donald S.; Stokes, Matthew P.; Vijapurkar, Ulka; Hatzivassiliou, Georgia; Friedman, Lori S.; Belvin, Marcia

doi:10.1002/pmic.201600118

Cited by 4 publications

(5 citation statements)

References 29 publications

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“…This was an expected observation since previous studies have reported a similar phosphotyrosine enrichment profile using metal ion enrichment chromatography despite significant induction of cellular tyrosine phosphorylation, for instance, via pervanadate treatment [ 34 , 35 ]. Additionally, singly phosphorylated peptides were more strongly represented than doubly or triply phosphorylated peptides in our dataset, consistent with previous reports [ 33 , 36 , 37 ] (Fig. 2d ).…”

Section: Resultssupporting

confidence: 93%

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

et al. 2018

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BackgroundThe non-receptor tyrosine kinase, SRMS (Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) is a member of the BRK family kinases (BFKs) which represents an evolutionarily conserved relative of the Src family kinases (SFKs). Tyrosine kinases are known to regulate a number of cellular processes and pathways via phosphorylating substrate proteins directly and/or by partaking in signaling cross-talks leading to the indirect modulation of various signaling intermediates. In a previous study, we profiled the tyrosine-phosphoproteome of SRMS and identified multiple candidate substrates of the kinase. The broader cellular signaling intermediates of SRMS are unknown.MethodsIn order to uncover the broader SRMS-regulated phosphoproteome and identify the SRMS-regulated indirect signaling intermediates, we performed label-free global phosphoproteomics analysis on cells expressing wild-type SRMS. Using computational database searching and bioinformatics analyses we characterized the dataset.ResultsOur analyses identified 60 hyperphosphorylated (phosphoserine/phosphothreonine) proteins mapped from 140 hyperphosphorylated peptides. Bioinfomatics analyses identified a number of significantly enriched biological and cellular processes among which DNA repair pathways were found to be upregulated while apoptotic pathways were found to be downregulated. Analyses of motifs derived from the upregulated phosphosites identified Casein kinase 2 alpha (CK2α) as one of the major potential kinases contributing to the SRMS-dependent indirect regulation of signaling intermediates.ConclusionsOverall, our phosphoproteomics analyses identified serine/threonine phosphorylation dynamics as important secondary events of the SRMS-regulated phosphoproteome with implications in the regulation of cellular and biological processes.Electronic supplementary materialThe online version of this article (10.1186/s12953-018-0143-7) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 93%

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

et al. 2018

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“…Future work will be important to more fully dissect the interaction between NT5C and the Arp2/3 complex and its role in PI3K-regulated cell motility. Our study adds to the growing realization that PI3K pathway activation in cells leads to an extensive remodelling of the transcriptome and proteome46525354, a phenotype referred to as a ‘butterfly effect’5355.…”

Section: Discussionmentioning

confidence: 78%

Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate

Moniz

Šurinová

Ghazaly

et al. 2017

Sci Rep

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To identify novel effectors and processes regulated by PI3K pathway activation, we performed an unbiased phosphoproteomic screen comparing two common events of PI3K deregulation in cancer: oncogenic Pik3ca mutation (Pik3caH1047R) and deletion of Pten. Using mouse embryonic fibroblast (MEF) models that generate inducible, low-level pathway activation as observed in cancer, we quantified 7566 unique phosphopeptides from 3279 proteins. A number of proteins were found to be differentially-regulated by Pik3caH1047R and Pten loss, suggesting unique roles for these two events in processes such as vesicular trafficking, DNA damage repair and RNA splicing. We also identified novel PI3K effectors that were commonly-regulated, including putative AKT substrates. Validation of one of these hits, confirmed NT5C (5′,3′-Nucleotidase, Cytosolic) as a novel AKT substrate, with an unexpected role in actin cytoskeleton regulation via an interaction with the ARP2/3 complex. This study has produced a comprehensive data resource and identified a new link between PI3K pathway activation and actin regulation.

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“…One of the first phosphoproteomics studies intensively monitored phosphorylation dynamics in HeLa cells upon treatment with Epidermal Growth Factor (EGF) already in 2006 [9]. Phosphoproteomics has also been used to study various oncogenic signaling pathways such as Ras signaling [10] and PI3K signaling [11]. It has been applied to study lung cancer [12,13], ovarian cancer [14], neuroblastoma [15,16], and many other cancer entities.…”

Section: Introductionmentioning

confidence: 99%

Phosphoproteomics of short-term hedgehog signaling in human medulloblastoma cells

et al. 2020

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Background: Aberrant hedgehog (HH) signaling is implicated in the development of various cancer entities such as medulloblastoma. Activation of GLI transcription factors was revealed as the driving force upon pathway activation. Increased phosphorylation of essential effectors such as Smoothened (SMO) and GLI proteins by kinases including Protein Kinase A, Casein Kinase 1, and Glycogen Synthase Kinase 3 β controls effector activity, stability and processing. However, a deeper and more comprehensive understanding of phosphorylation in the signal transduction remains unclear, particularly during early response processes involved in SMO activation and preceding GLI target gene regulation. Methods: We applied temporal quantitative phosphoproteomics to reveal phosphorylation dynamics underlying the short-term chemical activation and inhibition of early hedgehog signaling in HH responsive human medulloblastoma cells. Medulloblastoma cells were treated for 5.0 and 15 min with Smoothened Agonist (SAG) to induce and with vismodegib to inhibit the HH pathway. Results: Our phosphoproteomic profiling resulted in the quantification of 7700 and 10,000 phosphosites after 5.0 and 15 min treatment, respectively. The data suggest a central role of phosphorylation in the regulation of ciliary assembly, trafficking, and signal transduction already after 5.0 min treatment. ERK/MAPK signaling, besides Protein Kinase A signaling and mTOR signaling, were differentially regulated after short-term treatment. Activation of Polo-like Kinase 1 and inhibition of Casein Kinase 2A1 were characteristic for vismodegib treatment, while SAG treatment induced Aurora Kinase A activity. Distinctive phosphorylation of central players of HH signaling such as SMO, SUFU, GLI2 and GLI3 was observed only after 15 min treatment.

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Quantitative phosphoproteomic analysis of the PI3K-regulated signaling network

Cited by 4 publications

References 29 publications

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

Global phosphoproteomic analysis identifies SRMS-regulated secondary signaling intermediates

Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate

Phosphoproteomics of short-term hedgehog signaling in human medulloblastoma cells

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