Abstract
In addition to its well-known glycolytic activity, GAPDH displays multiple functions, such as nuclear RNA export, DNA replication and repair, and apoptotic cell death. This functional diversity depends on its intracellular localization. In this study, we explored the signal transduction pathways involved in the nuclear translocation of GAPDH using confocal laser scanning microscopy of immunostained human diploid fibroblasts (HDFs). GAPDH was present mainly in the cytoplasm when cultured with 10% FBS. Serum depletion by culturing cells in a serum-free medium (SFM) led to a gradual accumulation of GAPDH in the nucleus, and this nuclear accumulation was reversed by the re-addition of serum or growth factors, such as PDGF and lysophosphatidic acid. The nuclear export induced by the re-addition of serum or growth factors was prevented by LY 294002 and SH-5, inhibitors of phosphoinositide 3-kinase (PI3K) and Akt/protein kinase B, respectively, suggesting an involvement of the PI3K signaling pathway in the nuclear export of GAPDH. In addition, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulated the nuclear translocation of GAPDH and prevented serum- and growth factor-induced GAPDH export. AMPK inhibition by compound C or AMPK depletion by siRNA treatment partially prevented SFM- and AICAR-induced nuclear translocation of GAPDH. Our data suggest that the nuclear translocation of GAPDH might be regulated by the PI3K signaling pathway acting mainly as a nuclear export signal and the AMPK signaling pathway acting as a nuclear import signal.
Highlights
GAPDH (E.C. 1.2.1.12) functions as a glycolytic enzyme working within the cytoplasm
Following serum depletion and the re-addition of serum, PDGF, and lysophosphatidic acid (LPA), we examined the intracellular distribution of GAPDH by confocal laser scanning microscopy of immunostained human diploid fibroblasts (HDFs)
Because serum depletion had no influence on the total GAPDH expression level, we investigated the intracellular distribution of GAPDH by confocal laser scanning microscopy of immunostained HDFs (Figures 1A and 1B)
Summary
GAPDH (E.C. 1.2.1.12) functions as a glycolytic enzyme working within the cytoplasm. GAPDH forms a tetramer to be active and catalyzes the oxidative phosphorylation of glyceraldehyde 3-phosphate, producing 1,3-bisphosphoglycerate. Besides its role in glycolysis, GAPDH is involved in several biological functions, including the organization of the cytoskeleton and regulation of endocytosis, binding and transport of tRNA, and regulation of translation, transcription, replication, DNA repair, cell proliferation, and apoptosis (Sirover, 2005; Harada et al, 2007). Analysis of the growth factor signaling pathway with specific inhibitors has revealed that the nuclear export of GAPDH is prevented by LY 294002, an inhibitor of phosphoinositide 3-kinase (PI3K) (Schmitz, 2001). Activated PI3K produces phosphatidyl inositol 3,4,5-triphosphate (PIP3), which interacts with a variety of molecules, such as Akt kinase ( called PKB) via pleckstrin homology (PH) domains of these downstream targets (Cooray, 2004). Akt is a serine/threonine kinase that phosphorylates many different target proteins including GSK-3β, Bad, caspase-9, forkhead transcription factors and NF-kB (Cooray, 2004)
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