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Mol. Cells 2008; 26(2): 200-205

Published online January 1, 1970

© The Korean Society for Molecular and Cellular Biology

Analysis of Dual Phosphorylation of Hog1 MAP Kinase in Saccharomyces cerevisiae Using Quantitative Mass Spectrometry

Min-Yeon Choi, Gum-Yong Kang, Jae-Young Hur, Jin Woo Jung, Kwang Pyo Kim and Sang-Hyun Park

Abstract

The mitogen-activated protein kinase (MAPK) signaling pathway is activated in response to extracellular stimuli and regulates various activities in eukaryotic cells. Following exposure to stimuli, MAPK is known to be activated via dual phosphorylation at a conserved TxY motif in the activation loop; both threonine and tyrosine residues are phosphorylated by an upstream kinase. However, the mechanism underlying dual phosphorylation is not clearly understood. In the budding yeast Saccharomyces cerevisiae, the Hog1 MAPK mediates the high-osmolarity glycerol (HOG) signaling pathway. Tandem mass spectrometry and phosphospecific immunoblotting were performed to quantitatively monitor the dynamic changes occurring in the phosphorylation status of the TxY motif of Hog1 on exposure to osmotic stress. The results of our study suggest that the tyrosine residue is preferentially and dynamically phosphorylated following stimulation, and this in turn leads to the dual phosphorylation. The tyrosine residue was hyperphosphorylated in the absence of a threonine residue; this result suggests that the threonine residue is critical for the control of signaling noise and adaptation to osmotic stress.

Keywords Hog1, MAP kinase, mass spectrometry, phosphorylation, MS/MS, signal transduction, yeast

Article

Research Article

Mol. Cells 2008; 26(2): 200-205

Published online August 31, 2008

Copyright © The Korean Society for Molecular and Cellular Biology.

Analysis of Dual Phosphorylation of Hog1 MAP Kinase in Saccharomyces cerevisiae Using Quantitative Mass Spectrometry

Min-Yeon Choi, Gum-Yong Kang, Jae-Young Hur, Jin Woo Jung, Kwang Pyo Kim and Sang-Hyun Park

Abstract

The mitogen-activated protein kinase (MAPK) signaling pathway is activated in response to extracellular stimuli and regulates various activities in eukaryotic cells. Following exposure to stimuli, MAPK is known to be activated via dual phosphorylation at a conserved TxY motif in the activation loop; both threonine and tyrosine residues are phosphorylated by an upstream kinase. However, the mechanism underlying dual phosphorylation is not clearly understood. In the budding yeast Saccharomyces cerevisiae, the Hog1 MAPK mediates the high-osmolarity glycerol (HOG) signaling pathway. Tandem mass spectrometry and phosphospecific immunoblotting were performed to quantitatively monitor the dynamic changes occurring in the phosphorylation status of the TxY motif of Hog1 on exposure to osmotic stress. The results of our study suggest that the tyrosine residue is preferentially and dynamically phosphorylated following stimulation, and this in turn leads to the dual phosphorylation. The tyrosine residue was hyperphosphorylated in the absence of a threonine residue; this result suggests that the threonine residue is critical for the control of signaling noise and adaptation to osmotic stress.

Keywords: Hog1, MAP kinase, mass spectrometry, phosphorylation, MS/MS, signal transduction, yeast

Mol. Cells
Jun 30, 2023 Vol.46 No.6, pp. 329~398
COVER PICTURE
The cellular proteostasis network is adaptively modulated upon cellular stress, thereby protecting cells from proteostasis collapse. Heat shock induces the translocation of misfolded proteins and the chaperone protein HSP70 into nucleolus, where nuclear protein quality control primarily occurs. Nuclear RNA export factor 1 (green), nucleolar protein fibrillarin (red), and nuclei (blue) were visualized in NIH3T3 cells under basal (left) and heat shock (right) conditions (Park et al., pp. 374-386).

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