Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Biol Chem. Nov 26, 2015; 6(4): 366-378
Published online Nov 26, 2015. doi: 10.4331/wjbc.v6.i4.366
Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions
Manali Phadke, Natalia Krynetskaia, Anurag Mishra, Carlos Barrero, Salim Merali, Scott A Gothe, Evgeny Krynetskiy
Manali Phadke, Natalia Krynetskaia, Carlos Barrero, Salim Merali, Scott A Gothe, Evgeny Krynetskiy, Temple University School of Pharmacy, Philadelphia, PA 19140, United States
Natalia Krynetskaia, Anurag Mishra, Evgeny Krynetskiy, Jayne Haines Center for Pharmacogenomics and Drug Safety, Temple University, Philadelphia, PA 19140, United States
Author contributions: Krynetskaia N and Krynetskiy E designed the research, analyzed and evaluated data; Phadke M, Mishra A and Barrero C performed the experiments and prepared corresponding sections for the manuscript; Merali S analyzed MS data; Gothe SA performed molecular modeling and data evaluation; all authors drafted the article and made critical revisions related to the intellectual content of the manuscript, and approved the final version of the article to be published.
Supported by The National Cancer Institute, No. R01-CA104729; Jayne Haines Center for Pharmacogenomics and Drug Safety of Temple University School of Pharmacy and Temple University Summer Research Award (to Evgeny Krynetskiy).
Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Evgeny Krynetskiy, PhD, DSc, Associate Professor, Temple University School of Pharmacy, 3307 North Broad Street, Philadelphia, PA 19140, United States. ekrynets@temple.edu
Telephone: +1-215-7074257 Fax: +1-215-7073678
Received: April 29, 2015
Peer-review started: April 30, 2015
First decision: August 20, 2015
Revised: September 1, 2015
Accepted: September 29, 2015
Article in press: September 30, 2015
Published online: November 26, 2015
Processing time: 208 Days and 8.2 Hours
Abstract

AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents.

METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding.

RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions.

CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners.

Keywords: NAD+; Binding site; Fluorescence recovery after photobleaching; Nuclear proteins; Mutation; Glyceraldehyde 3-phosphate dehydrogenase; Anticancer agents

Core tip: We detected the phosphorylated amino acid residues Y94, S98, T99 within the NAD+ binding center of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Substitution of these amino acids with non-phosphorylated alanine residues did not abrogate intranuclear localization of GAPDH. Instead, such mutations altered the molecular dynamics parameters of intranuclear GAPDH probably by hindering its interactions with yet to be identified nuclear biomolecules. Our molecular modeling experiments invoke an important structural feature -T99-E97 H-bond likely involved in stabilization of NAD+ binding center.