Determination of protein phosphorylation by polyacrylamide gel electrophoresis

Lee, Chang-Ro; Park, Young-Ha; Min, Huitae; Kim, Yeon-Ran; Seok, Yeong‐Jae

doi:10.1007/s12275-019-9021-y

Cited by 43 publications

(32 citation statements)

References 43 publications

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“…2, A-D). It is well-known that altered mobility of protein species in SDS-PAGE can be attributed to post-translational modifications such as phosphorylation (55,56). This raised the possibility that A3B may be phosphorylated by CDK4.…”

Section: Cdk4 Does Not Affect A3b Phosphorylation Localization or Dmentioning

confidence: 99%

The DNA deaminase APOBEC3B interacts with the cell-cycle protein CDK4 and disrupts CDK4-mediated nuclear import of Cyclin D1

McCann

Klein

Leland

et al. 2019

Journal of Biological Chemistry

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Edited by Charles E. Samuel Apolipoprotein B mRNA editing enzyme catalytic subunitlike protein 3B (APOBEC3B or A3B), as other APOBEC3 members, is a single-stranded (ss)DNA cytosine deaminase with antiviral activity. A3B is also overexpressed in multiple tumor types, such as carcinomas of the bladder, cervix, lung, head/neck, and breast. A3B generates both dispersed and clustered C-toT and C-to-G mutations in intrinsically preferred trinucleotide motifs (TCA/TCG/TCT). A3B-catalyzed mutations are likely to promote tumor evolution and cancer progression and, as such, are associated with poor clinical outcomes. However, little is known about cellular processes that regulate A3B. Here, we used a proteomics approach involving affinity purification coupled to MS with human 293T cells to identify cellular proteins that interact with A3B. This approach revealed a specific interaction with cyclin-dependent kinase 4 (CDK4). We validated and mapped this interaction by co-immunoprecipitation experiments. Functional studies and immunofluorescence microscopy experiments in multiple cell lines revealed that A3B is not a substrate for CDK4-Cyclin D1 phosphorylation nor is its deaminase activity modulated. Instead, we found that A3B is capable of disrupting the CDK4-dependent nuclear import of Cyclin D1. We propose that this interaction may favor a more potent antiviral response and simultaneously facilitate cancer mutagenesis.

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Section: Cdk4 Does Not Affect A3b Phosphorylation Localization or Dmentioning

confidence: 99%

The DNA deaminase APOBEC3B interacts with the cell-cycle protein CDK4 and disrupts CDK4-mediated nuclear import of Cyclin D1

McCann

Klein

Leland

et al. 2019

Journal of Biological Chemistry

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“…Since VC1710 band was detected in the two fractions incubated with EIIA Glc and either glucose or PEP, we assumed that VC1710 interacts with both dephosphorylated and phosphorylated EIIA Glc . To validate the ligand-fishing data, His-EIIA Glc , EI, and HPr was mixed with glucose or PEP, and the phosphorylation state of His-EIIA Glc was confirmed by its mobility shift in an SDS–PAGE gel first 33 . Then a mixture of E. coli cell extracts expressing recombinant VC1710 and VcFapA (lane CE) was added and subjected to protein affinity pull-down assays to determine their interaction with either form of EIIA Glc (lanes 2 and 3 in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The phosphorylation state of EIIA Glc was determined as described previously 12,33 with some modifications. A 0.2-ml aliquot of cell culture was quenched at OD 600 ~ 0.5 by adding 20 μl of 5 M NaOH followed by vortexing for 15 s, and then 150 μl of 3 M sodium acetate (pH 5.3) and 0.9 ml of ethanol were sequentially added.…”

Section: Methodsmentioning

confidence: 99%

Sugar-mediated regulation of a c-di-GMP phosphodiesterase in Vibrio cholerae

et al. 2019

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Biofilm formation protects bacteria from stresses including antibiotics and host immune responses. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae, but the underlying mechanisms remain unclear. Here, we show that EIIAGlc, a component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), regulates the intracellular concentration of the cyclic dinucleotide c-di-GMP, and thus biofilm formation. The availability of preferred sugars such as glucose affects EIIAGlc phosphorylation state, which in turn modulates the interaction of EIIAGlc with a c-di-GMP phosphodiesterase (hereafter referred to as PdeS). In a Drosophila model of V. cholerae infection, sugars in the host diet regulate gut colonization in a manner dependent on the PdeS-EIIAGlc interaction. Our results shed light into the mechanisms by which some nutrients regulate biofilm formation and host colonization.

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“…When the molecular weight is different from what is expected that molecular weight is also important because it is usually associated with post-translational modifications of the target protein. Protein modifications like phosphorylation can increase the molecular weight of a protein during electrophoresis (16). Glycosylated protein content when heterogeneously present among the proteins can be responsible for high variations in protein mobility through electrophoresis.…”

Section: Discussionmentioning

confidence: 99%

Incorrect Molecular Weights due to inaccurate Prestained Protein Molecular Weight Markers that are used for Gel Electrophoresis and Western Blotting

Neris¹,

Kaur²,

Gomes³

2020

Preprint

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The most widely used Western blotting protein standards are prestained proteins of known molecular mass (kDa). They are also utilized for sodium dodecyl sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) to determine the molecular mass of proteins separated by electrophoresis. The objective of this study was to assess the reliability of different commercially available protein standards in predicting accurate protein molecular weights. We performed this experiment by running Criterion TGX gels with five prestained protein standards (Thermo Fisher SeeBlue Plus 2, Bio-Rad Precision Plus Protein Dual-color, Thermo Fisher Spectra Multi-color, Novex-Sharp Pre-stained, and Invitrogen iBright Pre-Stained). To evaluate their accuracy, we utilized highly purified Bovine Serum Albumin (BSA, 66.44 kDa) and Cytochrome C (Cyto C, 11.62 kDa). We also made use of the dimers of BSA (132.88 kDa) and Cyt C (23.24 kDa) that are present on SDS-PAGE gels. Our results suggest that three of the standards were less accurate at higher molecular masses with the iBright marker having the highest error in determining the expected 132.88 kDa molecular weight. The SeeBlue Plus 2 was accurate at identifying the 132.88 kDa molecular weight protein band but was less reliable for the three other lower molecular weight proteins. These findings have significant implications for the determination of protein masses because researchers rely on these standards to evaluate the molecular masses of their protein(s).We suggest that at least two different protein standards should be initially used in electrophoresis gels and for Western blotting in order to get accurate protein molecular weight results.

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Determination of protein phosphorylation by polyacrylamide gel electrophoresis

Cited by 43 publications

References 43 publications

The DNA deaminase APOBEC3B interacts with the cell-cycle protein CDK4 and disrupts CDK4-mediated nuclear import of Cyclin D1

The DNA deaminase APOBEC3B interacts with the cell-cycle protein CDK4 and disrupts CDK4-mediated nuclear import of Cyclin D1

Sugar-mediated regulation of a c-di-GMP phosphodiesterase in Vibrio cholerae

Incorrect Molecular Weights due to inaccurate Prestained Protein Molecular Weight Markers that are used for Gel Electrophoresis and Western Blotting

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