Analysis of dual phosphorylation of Hog1 MAP kinase in Saccharomyces cerevisiae using quantitative mass spectrometry.
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.
Choi
Min-Yeon
MY
Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Kang
Gum-Yong
GY
Hur
Jae-Young
JY
Jung
Jin Woo
JW
Kim
Kwang Pyo
KP
Park
Sang-Hyun
SH
D000595:Amino Acid Sequence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009997:Osmotic Pressure; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D029701:Saccharomyces cerevisiae Proteins; D013912:Threonine; D014443:Tyrosine; C088589:HOG1 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases