Abstract
Sufficiency and depletion of nutrients regulate the cellular activities through the protein phosphorylation reaction; however, many protein substrates remain to be clarified. GBF1 (Golgi-specific brefeldin A resistance factor 1), a guanine nucleotide exchange factor for the ADP-ribosylation factor family associated with the Golgi apparatus, was isolated as a phosphoprotein from the glucose-depleted cells by using the phospho-Akt-substrate antibody, which recognizes the substrate proteins of several protein kinases. The phosphorylation of GBF1 was induced by 2-deoxyglucose (2-DG), which blocks glucose utilization and increases the intracellular AMP concentration, and by AICAR, an AMP-activated protein kinase (AMPK) activator. This phosphorylation was observed in the cells expressing the constitutively active AMPK. The 2-DG-induced phosphorylation of GBF1 was suppressed by Compound C, an AMPK inhibitor, and by the overexpression of the kinase-negative AMPK. Analysis using the deletion and point mutants identified Thr(1337) as the 2-DG-induced phosphorylation site in GBF1, which is phosphorylated by AMPK in vitro. ATP depletion is known to provoke the Golgi apparatus disassembly. Immunofluorescent microscopic analysis with the Golgi markers indicated that GBF1 associates with the fragmented Golgi apparatus in the cells treated with 2-DG and AICAR. The expression of the kinase-negative AMPK and the GBF1 mutant replacing Thr(1337) by Ala prevented the 2-DG-induced Golgi disassembly. These results indicate that GBF1 is a novel AMPK substrate and that the AMPK-mediated phosphorylation of GBF1 at Thr(1337) has a critical role, presumably by attenuating the function of GBF1, in the disassembly of the Golgi apparatus induced under stress conditions that lower the intracellular ATP concentration.
Highlights
Lular activities through the protein phosphorylation reaction, as hormones and growth factors regulate the biological functions by the membrane receptor stimulation and the following protein phosphorylation cascades [1,2,3]
Peptide mass fingerprinting analysis using mass spectrometry identified p500 as plectin, a linker protein that can associate with the cytoskeletal filament networks and many membrane proteins [23], and identified p200 as GBF1, which was originally identified as a Golgi apparatus-associated guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family containing a SEC7 domain for the GEF activity [24] (Fig. 1B)
We employed the PAS antibody for the screening of the phosphoproteins produced by the nutrient signals, and GBF1 was isolated, which was revealed to be a substrate of AMPK
Summary
Lular activities through the protein phosphorylation reaction, as hormones and growth factors regulate the biological functions by the membrane receptor stimulation and the following protein phosphorylation cascades [1,2,3]. Identification of GBF1 as a Nutrient-regulated Phosphoprotein—The screening of the phosphoproteins in the CHO-IR cell lysates by immunoprecipitation using the PAS antibody isolated two distinct proteins designated as p500 and p200 from the cells cultured in the media containing and deprived of nutrients (i.e. amino acids and glucose, respectively), as visualized by silver staining after SDS-PAGE (Fig. 1A, top).
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