Methods Applied to the Study of Protein Arginine Methylation

Cheng, Donghang; Vemulapalli, Vidyasiri; Bedford, Mark T.

doi:10.1016/b978-0-12-391940-3.00004-4

Cited by 29 publications

(31 citation statements)

References 56 publications

(61 reference statements)

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“…This is a significant improvement over previous methods that are time consuming and identify few sites. A recent review summarizes some of these methods (52). A typical strategy usually begins by demonstrating that a recombinant PRMT or PKMT can methylate a recombinant protein substrate in vitro and then uses deletion mapping to identify the methylation regions.…”

Section: Discussionmentioning

confidence: 99%

Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation

Guo

Zhou

et al. 2014

Molecular & Cellular Proteomics

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Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.

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Section: Discussionmentioning

confidence: 99%

Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation

Guo

Zhou

et al. 2014

Molecular & Cellular Proteomics

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403

445

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“…After extensive washes with GST binding buffer, the interacting proteins were separated by SDS-PAGE, and Western blotting was performed with HA-HRP or FLAG-HRP antibodies. Methylation of GST-GPS2 and other substrates was performed as described previously (27). Briefly, 1 g of substrate was incubated with 1 l of S-adenosyl-L-[methyl-3H]methionine (PerkinElmer Life Sciences) and 0.5 g of purified recombinant enzyme for 1 h at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Exchange Factor TBL1 and Arginine Methyltransferase PRMT6 Cooperate in Protecting G Protein Pathway Suppressor 2 (GPS2) from Proteasomal Degradation

Huang

Cardamone

Johnson

et al. 2015

Journal of Biological Chemistry

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Background: GPS2 is a multifunctional protein controlling cellular homeostasis, inflammation, and lipid metabolism. Results: Arginine methylation modulates GPS2 interaction with TBL1 and prevents its degradation upon Siah2 ubiquitination. Conclusion: A tightly regulated balance between stabilization and degradation determines GPS2 levels. Significance: Understanding the molecular mechanisms controlling GPS2 expression and localization is critical for dissecting its multiple roles in the cell.

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“…Phosphorylated at Its C Terminus-Most arginine methyltransferases display robust intrinsic enzymatic activity in in vitro methylation assays (27), suggesting that in a cellular context, this enzymatic activity must be regulated, possibly by PTM of the PRMT enzymes themselves. To test this hypothesis, we scanned the amino acid sequences of the nine PRMTs using Web-based software that predicted the kinase-specific phosphorylation sites (Scansite and NetPhorest) (28,29).…”

Section: Prmt5 Ismentioning

confidence: 99%

PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch

Espejo

Gao

Black

et al. 2017

Journal of Biological Chemistry

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Edited by John M. DenuPRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum-and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/ 14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.Arginine methylation is a common PTM 3 that alters roughly 0.5% of all arginine residues in the cells. There are three types of arginine methylation: monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine (1). PRMT5 is one of nine PRMTs, and it is responsible for the vast majority (Ͼ95%) of the symmetric dimethylarginine modifications (2). PRMT5 was first characterized as a transcriptional repressor for cyclin E1 (3), and in this context, it methylates histone H3R8me2s, H2AR3me2s, and H4R3me2s (4). An epigenetic silencing role for PRMT5 has also recently been reported for the cell cycle inhibitor p21 (5).However, PRMT5 clearly has a number of non-histone substrates that are localized to the cytoplasm and the plasma membrane (6). In the cytoplasm, PRMT5 forms part of the methylosome and methylates a number of splicing factors (7). In keeping with these observations, the conditional deletion of PRMT5 in neural stem cells leads to defects in the core splicing machinery, reduced constitutive splicing, and massive alterations in alternative splicing profiles (8). Thus, this arginine methyltransferase has key biological roles that are associated with each of the major cellular compartments (the nucleus, the cytoplasm, and the plasma membrane), although little is known about how the activity and localization of PRMT5 in these different compartments are regulated.There is an emerging interest in establishing how signal transduction pathways communicate with chromatin and regulate changes to the epigenetic landscapes (9). It is likely that enzymes like PRMT5 may be marked by different ...

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Methods Applied to the Study of Protein Arginine Methylation

Cited by 29 publications

References 56 publications

Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation

Immunoaffinity Enrichment and Mass Spectrometry Analysis of Protein Methylation

Exchange Factor TBL1 and Arginine Methyltransferase PRMT6 Cooperate in Protecting G Protein Pathway Suppressor 2 (GPS2) from Proteasomal Degradation

PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch

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