Establishment of an ectopically expressed and functional PRMT1 for proteomic analysis of arginine‐methylated proteins

Chang, Yuan I.; Lin, Sheng-Wei; Chiou, Yi Ying; Sung, Jung Sung; Cheng, Lee Chun; Lü, Yu; Sun, Kien-Wen; Chang, Keejong; Chao, Lin; Lin, Wey Jinq

doi:10.1002/elps.201000376

Cited by 5 publications

(10 citation statements)

References 25 publications

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“…It is also known that incapable of activating the Calreticulin exposure pathway is refractory to the anticancer therapies [31]. The upregulation of Calreticulin after 6-shogaol treatment in HeLa cells confirmed by immunoblotting analysis was in accordance with findings reporting the ability of curcumin to induce the unfolding protein response by upregulating the Calreticulin expression [32]. …”

Section: Discussionsupporting

confidence: 77%

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

Peng

et al. 2012

Evidence-Based Complementary and Alternative Medicine

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Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale). In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA) suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

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Section: Discussionsupporting

confidence: 77%

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

Peng

et al. 2012

Evidence-Based Complementary and Alternative Medicine

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“…The homogenates were centrifuged at 16,000× g for 60 min at 4°C, and supernatants were collected. To examine endogenous PRMT1 activity, cell homogenates (2 µg) were incubated with purified recombinant hnRNP A1, A2 or K proteins (2 µg), which are known substrates for PRMT1 [23], [24], [25], in 25 mM Tris-HCl, pH 8.0, and S -adenosyl-L-[ methyl - 3 H] methionine ( 3 H-AdoMet) at 30°C for 30 min. Reactions were stopped by the addition of SDS sample buffer and then subjected to SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Protein Arginine Methyltransferase 1 Interacts with and Activates p38α to Facilitate Erythroid Differentiation

Hua¹,

Chang²,

Yao

et al. 2013

PLoS ONE

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Protein arginine methylation is emerging as a pivotal posttranslational modification involved in regulating various cellular processes; however, its role in erythropoiesis is still elusive. Erythropoiesis generates circulating red blood cells which are vital for body activity. Deficiency in erythroid differentiation causes anemia which compromises the quality of life. Despite extensive studies, the molecular events regulating erythropoiesis are not fully understood. This study showed that the increase in protein arginine methyltransferase 1 (PRMT1) levels, via transfection or protein transduction, significantly promoted erythroid differentiation in the bipotent human K562 cell line as well as in human primary hematopoietic progenitor CD34+ cells. PRMT1 expression enhanced the production of hemoglobin and the erythroid surface marker glycophorin A, and also up-regulated several key transcription factors, GATA1, NF-E2 and EKLF, which are critical for lineage-specific differentiation. The shRNA-mediated knockdown of PRMT1 suppressed erythroid differentiation. The methyltransferase activity-deficient PRMT1G80R mutant failed to stimulate differentiation, indicating the requirement of arginine methylation of target proteins. Our results further showed that a specific isoform of p38 MAPK, p38α, promoted erythroid differentiation, whereas p38β did not play a role. The stimulation of erythroid differentiation by PRMT1 was diminished in p38α- but not p38β-knockdown cells. PRMT1 appeared to act upstream of p38α, since expression of p38α still promoted erythroid differentiation in PRMT1-knockdown cells, and expression of PRMT1 enhanced the activation of p38 MAPK. Importantly, we showed for the first time that PRMT1 was associated with p38α in cells by co-immunoprecipitation and that PRMT1 directly methylated p38α in in vitro methylation assays. Taken together, our findings unveil a link between PRMT1 and p38α in regulating the erythroid differentiation program and provide evidence suggesting a novel regulatory mechanism for p38α through arginine methylation.

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“…Fourteen proteins were identified from 18 spots as summarized in Table 1. Among these, eight proteins were previously reported as methylated proteins, including EF-2 [39], a-tubulin and -b [40], nucleolin [41], actin [42], hnRNP K [43], PPP2C [44], and vimentin [45]. Among the remaining proteins with putative methylation modifications, EIF4H contains a conserved RGG motif that is considered as the consensus arginine methylation site of substrates for PRMT1 (Supporting Information Fig.…”

Section: Identification Of Methylated Proteins Associated With V-src mentioning

confidence: 99%

“…Peptides were extracted with 0.1% TFA/50% ACN. An aliquot of extracted peptide was concentrated by SpeedVac (Thermo savant) for LC-MS/MS (Q-TOF2, Waters) analysis as described previously [33].…”

Section: In-gel Digestion and Ms Analysismentioning

confidence: 99%

“…Currently, direct identification of in vivo methylated proteins remains difficult due to the very limited choice of specific antibodies against different types of protein methylations. However, in vitro methylation of cell lysate in the presence of tritium-labeled methyl donors has been shown as an alternative approach to recognize the methylated proteins in mammalian cells [32,33]. Notably, in vitro methylation assay in diverse cell lysates could lead to differential methylation signals which may originate from the difference of enzyme activity/abundance or availability of methyl-accepting proteins.…”

Section: Introductionmentioning

confidence: 99%

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Proteomics analysis of in vitro protein methylation during Src‐induced transformation

Chiou¹,

Fu²,

Lin³

et al. 2012

Electrophoresis

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Src, a nonreceptor tyrosine kinase, was the first oncogene identified from an oncogenic virus. Mechanistic studies of Src-induced transformations aid in understanding the pathologic processes underlying tumorigenesis and may provide new strategies for cancer therapy. Although several pathways and protein modifications are reportedly involved in Src-induced transformation, the detailed mechanisms of their regulation remain unclear. Protein methylation is an important PTM that is widely involved in cellular physiology. In this study, we determined if protein methylation was involved in Src activation and which methylated proteins were associated with this activity. Using in vitro methylation and 2-DE analysis of viral Src (v-Src)-transformed rat kidney epithelial cells (RK3E), several known and novel methylated proteins were identified based on their changes in methylation signal intensity upon transformation. Among these, elongation factor 2 (EF-2), heterogeneous nuclear ribonucleoprotein K (hnRNP K), and β-tubulin protein expressions remained unchanged, indicating that their altered methylation levels were due to Src activation. In addition, the altered expression of β-actin, vimentin, and protein phosphatase 2, catalytic subunit (PPP2C) as well as protein phosphatase 2, catalytic subunit methylation were also confirmed in RK3E cells transformed with a human oncogenic Src mutant (Src531), supporting their association with Src-induced transformation in human cancer. Together, we showed putative involvement of protein methylation in Src activation and our identification of methylated proteins provides important targets for extensively studying Src-induced transformations.

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Establishment of an ectopically expressed and functional PRMT1 for proteomic analysis of arginine‐methylated proteins

Cited by 5 publications

References 25 publications

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

Protein Arginine Methyltransferase 1 Interacts with and Activates p38α to Facilitate Erythroid Differentiation

Proteomics analysis of in vitro protein methylation during Src‐induced transformation

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