Two-component signaling to the stress MAP kinase cascade in fission yeast.

Susumu Morigasaki, Kazuhiro Shiozaki

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

In the fission yeast Schizosaccharomyces pombe, the Mak2/3 sensor histidine kinases (HKs), the Mpr1 histidine-containing phosphotransfer (HPt) protein, and the Mcs4 response regulator (RR) constitute a multistep phosphorelay, which is connected to a stress-activated mitogen-activated protein kinase (MAPK) cascade. This hybrid signaling pathway senses H2O2 and transmits the stress signal by sequential phosphorylation of the component proteins, whose physical interactions play crucial roles to attain eventual activation of Spc1 MAPK. This chapter describes methodological details of the copurification assays in S. pombe cell lysate to detect the physical interactions between the Mpr1 HPt and Mcs4 RR proteins and between Mcs4 and the MAPK kinase kinases (MAPKKKs) of the Spc1 cascade. Unexpectedly, we found that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by tdh1+ is involved in the H2O2 signaling process, and its association with Mcs4 and MAPKKKs in cell lysate is also detectable by copurification assays. In response to H2O2, the catalytic cysteine residue of Tdh1 GAPDH is subjected to S-thiolation, of which detection protocol is described as well.

Original languageEnglish (US)
Pages (from-to)279-289
Number of pages11
JournalMethods in Enzymology
Volume471
StatePublished - 2010
Externally publishedYes

Fingerprint

Schizosaccharomyces
MAP Kinase Signaling System
Histidine
Yeast
Glyceraldehyde-3-Phosphate Dehydrogenases
Phosphotransferases
Mitogen-Activated Protein Kinases
Assays
MAP Kinase Kinase Kinases
Phosphorylation
Proteins
Mitogen-Activated Protein Kinase Kinases
Cysteine
Chemical activation
Association reactions
Sensors
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Two-component signaling to the stress MAP kinase cascade in fission yeast. / Morigasaki, Susumu; Shiozaki, Kazuhiro.

In: Methods in Enzymology, Vol. 471, 2010, p. 279-289.

Research output: Contribution to journalArticle

Morigasaki, Susumu ; Shiozaki, Kazuhiro. / Two-component signaling to the stress MAP kinase cascade in fission yeast. In: Methods in Enzymology. 2010 ; Vol. 471. pp. 279-289.
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