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Identification of autophosphorylationsites in recombinant Saccharomyces cerevisiae Ire1

ABSTRACT: This study reports autophosphorylation sites in the serine/threonine protein kinase Ire1. The cytosolic portion of Ire1 was expressed as an glutathione S transferase fusion protein in Escherichia coli. Phosphroyaltion sites in the recombinant wild type protein and a L745A mutant were mapped by mass spectrometry. Phosphorylation sites map of the activation loop in the protein kinase domain and the alphaEF-alphaF insertion loop. In the activation loop phosphorylation was observed at serine 837, serine 840, serine 844, and threonine 844. Phosphorylaton was also observed at serine 850 in the P+1 loop. In the alphaEF-alphaF insertion loop phosphorylation was observed at threonine 873, serine 877, serine 878, threonine 881, serine 884, serine 885, and serine 887.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Martin Schroeder

LAB HEAD: Martin Schröder

PROVIDER: PXD004924 | Pride | 2017-06-09

REPOSITORIES: Pride

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Bypass of Activation Loop Phosphorylation by Aspartate 836 in Activation of the Endoribonuclease Activity of Ire1.

Armstrong Michael C MC Šestak Sergej S Ali Ahmed A AA Sagini Hanan A M HAM Brown Max M Baty Karen K Treumann Achim A Schröder Martin M

Molecular and cellular biology 20170728 16

The bifunctional protein kinase-endoribonuclease Ire1 initiates splicing of the mRNA for the transcription factor Hac1 when unfolded proteins accumulate in the endoplasmic reticulum. Activation of Saccharomyces cerevisiae Ire1 coincides with autophosphorylation of its activation loop at S840, S841, T844, and S850. Mass spectrometric analysis of Ire1 expressed in Escherichia coli identified S837 as another potential phosphorylation site in vivo Mutation of all five potential ...[more]

PMID: 28559428

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