ArticleWorld journal of biological chemistry2015
Disruption of NAD(+) binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions.
Article in World journal of biological chemistry, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed, 10 citations in OpenAlex.
- Proteome and Metabolome Alterations in Radish (Biomolecules · 2025Article
- Biochemical evidence that the whole compartment activity behavior of GAPDH differs between the cytoplasm and nucleus.PloS one · 2023Article
- Age-related NADExperimental gerontology · 2020Review
- Proteome-Wide Characterization of Phosphorylation-Induced Conformational Changes in Breast Cancer.Journal of proteome research · 2018Article
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Authors and funding
7 authors at 1 institution in 1 country.
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Abstract
aimTo characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents.
methodsWe 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.
resultsUsing 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.
conclusionOur results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.
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