Quantitative profiling of dual phosphorylation of Fus3 MAP kinase in Saccharomyces cerevisiae.
Mitogen-activated protein kinase (MAPK) signaling is a crucial component of eukaryotic cells; it plays an important role in responses to extracellular stimuli and in the regulation of various cellular activities. The signaling cascade is evolutionarily conserved in the eukaryotic kingdom from yeast to human. In response to a variety of extracellular signals, MAPK activity is known to be regulated via phosphorylation of a conserved TxY motif at the activation loop in which both threonine and tyrosine residues are phosphorylated by the upstream kinase. However, the mechanism by which both residues are phosphorylated continues to remain elusive. In the budding yeast, Saccharomyces cerevisiae, Fus3 MAPK is involved in the mating signaling pathway. In order to elucidate the functional mechanism of MAPK activation, we quantitatively profiled phosphorylation of the TxY motif in Fus3 using mass spectrometry (MS). We used synthetic heavy stable isotope-labeled phosphopeptides and nonphosphopeptides corresponding to the proteolytic TxY motif of Fus3 and accompanying data-dependent tandem MS to quantitatively monitor dynamic changes in the phosphorylation events of MAPK. Phosphospecific immunoblotting and the MS data suggested that the tyrosine residue is dynamically phosphorylated upon stimulation and that this leads to dual phosphorylation. In contrast, the magnitude of threonine phosphorylation did not change significantly. However, the absence of a threonine residue leads to hyperphosphorylation of the tyrosine residue in the unstimulated condition, suggesting that the threonine residue contributes to the control of signaling noise.
Hur
Jae-Young
JY
Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Kang
Gum-Yong
GY
Choi
Min-Yeon
MY
Jung
Jin Woo
JW
Kim
Kwang-Pyo
KP
Park
Sang-Hyun
SH
D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D047468:Immunoprecipitation; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000378:metabolism
D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; C075580:FUS3 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases