Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1α
BMC Biochemistry
Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1
Daniel Itzhak 0 1
Michael Bright 0 1
Peter McAndrew 1
Amin Mirza 1
Yvette Newbatt 1
Jade Strover 1
Marcella Widya 2
Andrew Thompson 2
Gareth Morgan 1
Ian Collins 0
Faith Davies 0 1
0 Division of Molecular Pathology, Institute of Cancer Research , Sutton, Surrey SM2 5NG , UK
1 From the Division of Cancer Therapeutics, Institute of Cancer Research , Sutton, Surrey SM2 5NG , UK
2 Proteomics Core Facility, Institute of Cancer Research , London SW3 6JB , UK
Background: Endoplasmic reticulum stress, caused by the presence of misfolded proteins, activates the stress sensor inositol-requiring enzyme 1 (IRE1). The resulting increase in IRE1 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 IRE1 activation is currently unclear. The role of IRE1 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 IRE1 kinase activity using wild-type cytosolic protein constructs. Results: We show that concentration-dependent oligomerisation is sufficient to cause IRE1 cytosolic domain RNase activity in vitro. We demonstrate a role for the kinase activity by showing that autophosphorylation enhances RNase activity. Inclusion of the IRE1 linker domain in protein constructs allows hyperphosphorylation and further enhancement of RNase activity, highlighting the importance of kinase activity. We show that IRE1 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 IRE1 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 IRE1 RNase activity which is achieved by increasing the propensity of IRE1 to dimerise. This work supports the development of IRE1 kinase inhibitors for use in the treatment of secretory cancers.
Endoplasmic reticulum stress; Enzyme mechanisms; ER stress; Mass spectrometry (MS); Multiple myeloma; Ribonuclease; Unfolded protein response; IRE1; Autophosphorylation
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Background
Inositol-requiring enzyme 1 (IRE1) is an endoplasmic
reticulum (ER) stress sensor activated by the
accumulation of unfolded proteins. IRE1 activation results in the
production of XBP1s, a transcription factor, leading to
increased expression of genes involved in membrane
synthesis, protein folding and protein degradation [1-3],
termed the unfolded protein response (UPR) [4]. This
response enables cells to adapt to ER stress caused for
example by an increased protein load [5]. The UPR has
recently been shown to play an important role in cancer
biology, particularly in tumours with a secretory cell
origin [6,7]. An example of this is multiple myeloma, a
malignancy of plasma cells, which produce large quantities
of an immunoglobulin or paraprotein. These cells are
addicted to the UPR to manage the high protein
production which would otherwise be toxic. Thus, IRE1
activity and XBP1s production are thought to be critical to
the development and maintenance of the myeloma clone
[3,8,9] and have therefore been proposed as possible
therapeutic targets [10].
IRE1 consists of a lumenal stress-sensing domain,
transmembrane helix, cytosolic linker domain followed
by kinase and RNase domains [11]. Accumulation of
unfolded proteins in the ER lumen leads to release of binding
protein (BiP) from the IRE1 lumenal domain allowing
dimerisation [12]. In yeast, direct binding of unfolded
protein to Ire1 is additionally required for oligomerisation
[13] though this is not thought to occur with human
IRE1 [14]. The resulting oligomerisation enables
transautophosphorylation of the Ire1/IRE1 cytosolic domain
which activates the RNase [12,15], whose active site is
generated by dimerisation [16].
A number of pieces of data support a model where
oligomerisation is essential and kinase activity is
dispensable for the RNase activity. The requirement for
autophosphorylation in yeast and human IRE1 can be
made redundant by small molecules which bind the
kinase ATP site in lumenal domain deleted Ire1 or kinase
mutated Ire1 [17-19]. In vitro studies have also
demonstrated that inclusion of the Ire1 linker domain permits
the formation of higher-order oligomeric structures and
increased activity, even when the kinase is mutated [18].
Although the validity of this model is debated as kinase
inactivating mutations may or may not lack activity [20,21].
Moreover, the link (...truncated)