title: Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1α.
abstract: BACKGROUND
  Endoplasmic reticulum stress, caused by the presence of misfolded proteins, activates the stress sensor inositol-requiring enzyme 1α (IRE1α). The resulting increase in IRE1α RNase activity causes sequence-specific cleavage of X-box binding protein 1 (XBP1) mRNA, resulting in upregulation of the unfolded protein response and cellular adaptation to stress. The precise mechanism of human IRE1α activation is currently unclear. The role of IRE1α kinase activity is disputed, as results from the generation of various kinase-inactivating mutations in either yeast or human cells are discordant. Kinase activity can also be made redundant by small molecules which bind the ATP binding site. We set out to uncover a role for IRE1α kinase activity using wild-type cytosolic protein constructs.
  
  
  RESULTS
  We show that concentration-dependent oligomerisation is sufficient to cause IRE1α cytosolic domain RNase activity in vitro. We demonstrate a role for the kinase activity by showing that autophosphorylation enhances RNase activity. Inclusion of the IRE1α linker domain in protein constructs allows hyperphosphorylation and further enhancement of RNase activity, highlighting the importance of kinase activity. We show that IRE1α phosphorylation status correlates with an increased propensity to form oligomeric complexes and that forced dimerisation causes great enhancement in RNase activity. In addition we demonstrate that even when IRE1α is forced to dimerise, by a GST-tag, phospho-enhancement of activity is still observed.
  
  
  CONCLUSIONS
  Taken together these experiments support the hypothesis that phosphorylation is important in modulating IRE1α RNase activity which is achieved by increasing the propensity of IRE1α to dimerise. This work supports the development of IRE1α kinase inhibitors for use in the treatment of secretory cancers.
authors: Daniel
authors: D
authors: Itzhak
authors: Michael
authors: M
authors: Bright
authors: Peter
authors: P
authors: McAndrew
authors: Amin
authors: A
authors: Mirza
authors: Yvette
authors: Y
authors: Newbatt
authors: Jade
authors: J
authors: Strover
authors: Marcella
authors: M
authors: Widya
authors: Andrew
authors: A
authors: Thompson
authors: Gareth
authors: G
authors: Morgan
authors: Ian
authors: I
authors: Collins
authors: From the Division of Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK. faith.davies@icr.ac.uk.
authors: Faith
authors: F
authors: Davies
mesh_terms: D000595:Amino Acid Sequence; D059865:Endoplasmic Reticulum Stress; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases