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Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1α.

Itzhak D, Bright M, McAndrew P, Mirza A, Newbatt Y, Strover J, Widya M, Thompson A, Morgan G, Collins I, Davies F - BMC Biochem. (2014)

Bottom Line: 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.In addition we demonstrate that even when IRE1α is forced to dimerise, by a GST-tag, phospho-enhancement of activity is still observed.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: From the Division of Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK. faith.davies@icr.ac.uk.

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.

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Phosphorylation enhances activity of IRE1α in vitro. (A) Schematic of the truncated G547 and H499 IRE1α construct compared to the full-length protein. (B) Deconvoluted mass spectra of lambda phosphatase-treated G547 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of 3 phosphates due to autophosphorylation. (C) Schematic of the in silico designed stembulge RNA containing the XBP-1 splice site labelled 5’ with fluorescein (FAM) and 3’ with Black-Hole Quencher 1 (BHQ1) whose fluorescence quenching is alleviated upon cleavage. (D) 90 nM RNA in C was incubated with increasing concentrations of dephosphorylated IRE1α (open squares, EC50 = 369 nM ) or phosphorylated IRE1α (filled squares, EC50 = 114 nM) for 30 minutes at 30˚C. Error bars S.E.M of 3 independent experiments. (E) Linker regions of human and yeast IRE1. The linker domain is defined by the first residue after the transmembrane domain and the last residue before the kinase domain (human P465-S570, yeast Q556-L673). Human IRE1α linker domain is more Ser/Thr-rich 26/106aa (24.5%) than yeast Ire1 16/118aa (13.6%) linker domain. The lysine-rich region of the yeast linker domain is boxed. Full-length human IRE1α and yeast IRE1 sequences were aligned using EMBOSS stretcher [http://www.ebi.ac.uk/Tools/psa/emboss_stretcher/]. (F) Deconvoluted mass spectra of lambda phosphatase-treated H499 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of multiple phosphates (8–11) due to autophosphorylation. (G) As in D, dephosphorylated H499 IRE1α (open squares, EC50 = 440 nM), autophosphorylated H499 IRE1α (filled squares, EC50 = 77 nM).
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Figure 1: Phosphorylation enhances activity of IRE1α in vitro. (A) Schematic of the truncated G547 and H499 IRE1α construct compared to the full-length protein. (B) Deconvoluted mass spectra of lambda phosphatase-treated G547 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of 3 phosphates due to autophosphorylation. (C) Schematic of the in silico designed stembulge RNA containing the XBP-1 splice site labelled 5’ with fluorescein (FAM) and 3’ with Black-Hole Quencher 1 (BHQ1) whose fluorescence quenching is alleviated upon cleavage. (D) 90 nM RNA in C was incubated with increasing concentrations of dephosphorylated IRE1α (open squares, EC50 = 369 nM ) or phosphorylated IRE1α (filled squares, EC50 = 114 nM) for 30 minutes at 30˚C. Error bars S.E.M of 3 independent experiments. (E) Linker regions of human and yeast IRE1. The linker domain is defined by the first residue after the transmembrane domain and the last residue before the kinase domain (human P465-S570, yeast Q556-L673). Human IRE1α linker domain is more Ser/Thr-rich 26/106aa (24.5%) than yeast Ire1 16/118aa (13.6%) linker domain. The lysine-rich region of the yeast linker domain is boxed. Full-length human IRE1α and yeast IRE1 sequences were aligned using EMBOSS stretcher [http://www.ebi.ac.uk/Tools/psa/emboss_stretcher/]. (F) Deconvoluted mass spectra of lambda phosphatase-treated H499 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of multiple phosphates (8–11) due to autophosphorylation. (G) As in D, dephosphorylated H499 IRE1α (open squares, EC50 = 440 nM), autophosphorylated H499 IRE1α (filled squares, EC50 = 77 nM).

Mentions: An IRE1α kinase and RNase domain construct encompassing residues G547-L977, designated G547 IRE1α, which retains kinase autophosphorylation activity [24] was produced in insect cells (Figure 1A) and dephosphorylated by treatment with λ-phosphatase. Dephosphorylation was confirmed by western blot analysis using an antibody directed at phospho-serine 724 [24,25] (Additional file 1: Figure S1), and by mass spectrometry of intact protein (Figure 1B).


Multiple autophosphorylations significantly enhance the endoribonuclease activity of human inositol requiring enzyme 1α.

Itzhak D, Bright M, McAndrew P, Mirza A, Newbatt Y, Strover J, Widya M, Thompson A, Morgan G, Collins I, Davies F - BMC Biochem. (2014)

Phosphorylation enhances activity of IRE1α in vitro. (A) Schematic of the truncated G547 and H499 IRE1α construct compared to the full-length protein. (B) Deconvoluted mass spectra of lambda phosphatase-treated G547 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of 3 phosphates due to autophosphorylation. (C) Schematic of the in silico designed stembulge RNA containing the XBP-1 splice site labelled 5’ with fluorescein (FAM) and 3’ with Black-Hole Quencher 1 (BHQ1) whose fluorescence quenching is alleviated upon cleavage. (D) 90 nM RNA in C was incubated with increasing concentrations of dephosphorylated IRE1α (open squares, EC50 = 369 nM ) or phosphorylated IRE1α (filled squares, EC50 = 114 nM) for 30 minutes at 30˚C. Error bars S.E.M of 3 independent experiments. (E) Linker regions of human and yeast IRE1. The linker domain is defined by the first residue after the transmembrane domain and the last residue before the kinase domain (human P465-S570, yeast Q556-L673). Human IRE1α linker domain is more Ser/Thr-rich 26/106aa (24.5%) than yeast Ire1 16/118aa (13.6%) linker domain. The lysine-rich region of the yeast linker domain is boxed. Full-length human IRE1α and yeast IRE1 sequences were aligned using EMBOSS stretcher [http://www.ebi.ac.uk/Tools/psa/emboss_stretcher/]. (F) Deconvoluted mass spectra of lambda phosphatase-treated H499 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of multiple phosphates (8–11) due to autophosphorylation. (G) As in D, dephosphorylated H499 IRE1α (open squares, EC50 = 440 nM), autophosphorylated H499 IRE1α (filled squares, EC50 = 77 nM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3928614&req=5

Figure 1: Phosphorylation enhances activity of IRE1α in vitro. (A) Schematic of the truncated G547 and H499 IRE1α construct compared to the full-length protein. (B) Deconvoluted mass spectra of lambda phosphatase-treated G547 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of 3 phosphates due to autophosphorylation. (C) Schematic of the in silico designed stembulge RNA containing the XBP-1 splice site labelled 5’ with fluorescein (FAM) and 3’ with Black-Hole Quencher 1 (BHQ1) whose fluorescence quenching is alleviated upon cleavage. (D) 90 nM RNA in C was incubated with increasing concentrations of dephosphorylated IRE1α (open squares, EC50 = 369 nM ) or phosphorylated IRE1α (filled squares, EC50 = 114 nM) for 30 minutes at 30˚C. Error bars S.E.M of 3 independent experiments. (E) Linker regions of human and yeast IRE1. The linker domain is defined by the first residue after the transmembrane domain and the last residue before the kinase domain (human P465-S570, yeast Q556-L673). Human IRE1α linker domain is more Ser/Thr-rich 26/106aa (24.5%) than yeast Ire1 16/118aa (13.6%) linker domain. The lysine-rich region of the yeast linker domain is boxed. Full-length human IRE1α and yeast IRE1 sequences were aligned using EMBOSS stretcher [http://www.ebi.ac.uk/Tools/psa/emboss_stretcher/]. (F) Deconvoluted mass spectra of lambda phosphatase-treated H499 IRE1α produced in insect cells (grey) and after incubation with Mg/ATP in vitro (black) show the addition of multiple phosphates (8–11) due to autophosphorylation. (G) As in D, dephosphorylated H499 IRE1α (open squares, EC50 = 440 nM), autophosphorylated H499 IRE1α (filled squares, EC50 = 77 nM).
Mentions: An IRE1α kinase and RNase domain construct encompassing residues G547-L977, designated G547 IRE1α, which retains kinase autophosphorylation activity [24] was produced in insect cells (Figure 1A) and dephosphorylated by treatment with λ-phosphatase. Dephosphorylation was confirmed by western blot analysis using an antibody directed at phospho-serine 724 [24,25] (Additional file 1: Figure S1), and by mass spectrometry of intact protein (Figure 1B).

Bottom Line: 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.In addition we demonstrate that even when IRE1α is forced to dimerise, by a GST-tag, phospho-enhancement of activity is still observed.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: From the Division of Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK. faith.davies@icr.ac.uk.

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.

Show MeSH
Related in: MedlinePlus