Mutations in Ire1 kinase abolish phosphoryl-transfer but preserve RNase activity
Based on sequence conservation between Ire1 and related CDK2-like kinases as well as the recently solved crystal structures of the cytosolic portion of Ire1 (Lee et al., 2008
; Korennykh et al., 2009
), we designed an Ire1 variant with uncoupled kinase and RNase activities. To this end, we identified two catalytic residues, D797 and K799, in the nucleotide-binding pocket of Ire1 kinase. These residues are predicted to coordinate the terminal phosphate of ATP bound to Ire1 kinase (), and, by analogy to other kinases, are required to catalyze phosphotransfer (Lee et al., 2008
). We reasoned that mutating these residues to asparagines would preserve overall steric packing, hydrophobicity, and hydrogen bonding at the kinase-active site but disable proton transfer and thereby abolish phosphorylation (Fig. S1 A
). Thus, we expected that the mutant Ire1(D797N,K799N) would be kinase inactive but still able to activate its RNase via nucleotide binding.
Figure 1. Mutations in Ire1 kinase abolish phosphate transfer but preserve RNase activity. (A) A schematic representation of Ire1 depicting the location of each functional domain. Residues D797 and K799 in the nucleotide-binding pocket of the kinase domain hydrogen (more ...)
To carry out in vitro studies, we recombinantly expressed and purified the cytosolic portion of Ire1 WT and mutant Ire1. These constructs consisted of kinase and RNase domains preceded at the N terminus by 32 amino acids derived from the linker region that tethers the kinase domain to the transmembrane region. We previously showed that this peptide extension is important, as it enhances Ire1’s ability to activate its RNase by up to four orders of magnitude (Korennykh et al., 2009
). We term these constructs Ire1KR32 (WT) (Korennykh et al., 2009
) and Ire1KR32(D797N,K799N).
MALDI mass spectrometry analyses have shown that WT Ire1KR32 is highly phosphorylated when purified from Escherichia coli
, likely as a result of autophosphorylation (Korennykh et al., 2009
). Phosphorylation is evident in the mass-to-charge ratio (M/z) of WT Ire1KR32, which is higher than expected based on its theoretical molecular weight (Fig. S1 B). The shift of ~1.3 kD is consistent with the presence of ~17 phosphates and can be ameliorated by phosphatase treatment (Fig. S1 C). In contrast, purified Ire1KR32(D797N,K799N) has an M/z value that is precisely as expected based on its primary sequence, indicating that this protein is entirely unphosphorylated (Fig. S1 B; and see Fig. S7 in Korennykh et al., 2009
). These data suggest that Ire1KR32(D797N,K799N) is kinase inactive.
To confirm that Ire1KR32(D797N,K799N) was indeed kinase inactive, we measured trans-autophosphorylation of the recombinant proteins in an in vitro kinase assay. As expected, WT Ire1KR32 showed robust trans-autophosphorylation (, lanes 1–3) whereas Ire1KR32(D797N,K799N) exhibited no detectable kinase activity (, lanes 4–6). To show that the kinase-inactive Ire1 mutant is properly folded and is a competent substrate for phosphorylation, we mixed recombinant kinase-inactive Ire1 protein with a shorter WT version, Ire1KR, lacking the 32-amino acid peptide extension (Korennykh et al., 2009
). This enzyme retains WT kinase activity (, lanes 7–9) and can be distinguished from the Ire1KR32 versions by its lower molecular weight. When we mixed Ire1KR in vitro with Ire1KR32(D797N,K799N), we detected robust phosphorylation of the mutant enzyme (, lanes 10–12; top bands). In these mixing reactions, the top bands corresponding to the kinase-inactive variant of Ire1 were more extensively labeled with radioactive phosphate than WT enzyme. This is likely due to the greater number of unphosphorylated residues in kinase-inactive Ire1 available for phosphorylation when introduced to kinase-active enzyme.
Based on the previous observation that occupation of the active site of Ire1 kinase by nucleotide cofactor is sufficient to cause activation of the RNase, we expected that Ire1KR32(D797N,K799N) would retain RNase activity and that its activity would be stimulated by the presence of nucleotide. To test this prediction, we measured RNase activity in an in vitro cleavage assay using HP21, a previously characterized small substrate RNA containing a specific Ire1 cleavage site, in the presence or absence of ADP cofactor. In previous experiments, ADP stimulated Ire1KR32’s RNase activity by ~200-fold (Korennykh et al., 2009
). Here, in the absence of cofactor, both enzymes exhibited the same basal RNase activity as Ire1KR32 (, “APO”), consistent with previous observations (Korennykh et al., 2009
). Addition of ADP increased the RNase of Ire1KR32(D797N,K799N) 10-fold (versus ~100-fold for WT Ire1KR32; , “ADP”). These data are consistent with the idea that binding of cofactor stimulates the RNase activity of Ire1 in the absence of phosphorylation (Papa et al., 2003
). In in vitro assays using the HP21 substrate, the RNase activity of Ire1KR32(D797N,K799N) was 10-fold lower than that of WT. However, when a larger 443-nt Xbp1
mRNA-derived RNA fragment was used as a substrate (Korennykh et al., 2009
), Ire1KR32(D797N,K799N) cleaved with a rate (kobs
= 0.19 s−1
) indistinguishable from that of WT Ire1KR32 (kobs
= 0.19 s−1
; ). The Xbp1
mRNA is a 400-nt substrate derived from the mammalian counterpart to HAC1
mRNA. This substrate is cleaved by Ire1 in vitro with kinetics identical to that of HAC1
mRNA substrates of comparable length (unpublished data). The low ADP sensitivity of the Xbp1
mRNA cleavage reaction suggests a diminished requirement for cofactor during cleavage of this substrate. Present work in our laboratory is aimed at understanding the molecular mechanism of this phenomenon. This longer substrate RNA more closely resembles the endogenous in vivo substrate of Ire1 RNase, suggesting that kinase-inactive Ire1(D797N,K799N) should retain RNase function in living cells.
Ire1 kinase activity is dispensable for HAC1 mRNA splicing but enhances cell survival under ER stress
Because our in vitro results showed that we had successfully uncoupled the kinase and RNase functions of Ire1, we used kinase-inactive Ire1(D797N,K799N) to directly investigate the role of Ire1 kinase activity in vivo. This approach afforded the first opportunity to ask this question without requiring the addition of exogenous drug as past studies necessitated.
Our in vitro studies predict that cells expressing Ire1(D797N,K799N) should splice HAC1 mRNA upon UPR induction. To test this, we constructed a strain carrying a chromosomally integrated mutant IRE1 allele as the sole copy of IRE1 in the cell. We then induced the UPR and measured HAC1 mRNA splicing by Northern blotting. We induced ER stress with DTT, which causes protein misfolding in the ER by disrupting disulfide bond formation. As predicted, spliced HAC1 mRNA was produced upon DTT treatment in ire1(D797N,K799N) cells (, lanes 5 and 6). In contrast, HAC1 mRNA was not spliced in ire1Δ cells (, lanes 3 and 4). In these experiments, ire1(D797N,K799N) proved mildly hypomorphic, as the amount of HAC1 mRNA cleaved in the mutant cells was reduced compared with WT and HAC1 splicing intermediates were more abundant at the time point taken. This was not due to differences in the expression levels of Ire1 (). Nevertheless, these data reinforce the notion that Ire1 kinase activity is not required for RNA splicing.
Figure 2. Ire1 kinase activity, uncoupled from HAC1 mRNA splicing, is important for cell survival during the UPR. (A) Cells bearing WT IRE1 (lanes 1 and 2), a deletion of ire1 (lanes 3 and 4), or ire1(D797N,K799N) (lanes 5 and 6) were left uninduced (−) (more ...)
We were surprised to discover that splicing of HAC1 mRNA in ire1(D797N,K799N) cells failed to ensure cell survival under ER stress. When plated on medium containing tunicamycin, a drug that induces the UPR by blocking glycosylation in the ER, ire1(D797N,K799N) cells displayed a severe growth defect (). This resulted from loss of cell viability rather than growth arrest: sustained ER stress killed ire1(D797N,K799N) cells significantly earlier than WT cells ().
In search of an explanation for this growth defect, we tested whether functional Hac1 protein was produced from spliced HAC1
mRNA in ire1(D797N,K799N)
cells. To this end, we measured Hac1 protein production and determined the scope of the transcriptional response by assessing global mRNA expression after UPR induction. WT IRE1
cells expressing HA-tagged Hac1 were treated with DTT to induce the UPR and probed for HA-Hac1 by Western blotting. Ire1(D797N,K799N)
cells produced Hac1 protein at nearly WT levels (). Likewise, the microarray transcriptional profile of UPR-induced ire1(D797N,K799N)
cells revealed a profile nearly indistinguishable from that of WT cells (Fig. S2 A
). Canonical UPR target genes were up-regulated with similar kinetics, and to a comparable extent, in WT and ire1(D797N,K799N)
cells. Specific UPR target genes are highlighted in . Collectively, these data show that the observed reduction in HAC1
mRNA splicing in ire1(D797N,K799N)
cells does not lead to impairment of canonical UPR signaling.
Figure 3. Downstream events in UPR activation are normal in ire1(D797N,K799N) cells. (A) Microarray analysis was performed to assess the total mRNA expression profiles of WT IRE1 or ire1(D797N,K799N) cells over time after induction with 2 mM DTT. Cells were sampled (more ...)
One reason that a cell might die despite expression of target genes is that mRNAs are not translated. To confirm that protein products corresponding to UPR targets were also made, we determined Kar2 protein levels by Western blotting and measured global translation rates during the ER stress. The induction of Kar2 mirrored the microarray result for both WT and ire1(D797N,K799N) mutant cells (), confirming that expression of this canonical UPR target was intact in both strains. Furthermore, general translation rates were equivalent in both WT and ire1(D797N,K799N) cells (Fig. S2 B), indicating that global mRNA translation was not impaired in mutant cells. No explanation for the enhanced loss of cell viability of ire1(D797N,K799N) mutant cells was evident in these data.
As a consequence of UPR activation, the ER expands to meet the increased need for protein folding capacity (Cox et al., 1997
; Bernales et al., 2006
; Schuck et al., 2009
). To further ensure that UPR signaling downstream of Ire1 was unimpaired, we measured ER expansion. Using a GFP-tagged version of the ER marker Sec63 (Prinz et al., 2000
), we quantified expansion of the cortical ER before and after UPR induction in WT and ire1(D797N,K799N)
cells. In confocal sections through the middle of unstressed cells, the cortical ER marked by Sec63-GFP is visible underneath the plasma membrane as a broken line because the tubular ER network appears in cross section. Upon ER stress, the cortical ER is converted into expanded membrane sheets and appears as a continuous line. Consistent with microarray data showing normal induction of target genes, UPR-mediated ER expansion occurred normally in mutant cells (). Thus, the slight reduction in Hac1 protein produced in ire1(D797N,K799N)
cells () did not weaken UPR events downstream of Hac1 protein production. Collectively, the data presented thus far indicate that canonical UPR activation remains intact in ire1(D797N,K799N)
Ire1(D797N,K799N) fails to adapt to sustained ER stress
The homeostatic feedback response that is mediated by the UPR is characterized by an activation phase in which Ire1 begins to signal and an adaptive phase that occurs when cells adjust to ER stress and Ire1 is turned off (Pincus et al., 2010
). Because our findings indicate that Ire1 activation and induction of its downstream transcriptional targets are normal in ire1(D797N,K799N)
cells, we set out to examine the dynamics of Ire1 activation and attenuation in ire1(D797N,K799N)
cells. To this end, we took advantage of a splicing reporter, termed SR, previously developed in our laboratory (Aragón et al., 2009
). In the SR, the HAC1
ORF has been replaced by that of GFP
(), while the intron as well as the 5′ and 3′ untranslated regions (UTRs) of the HAC1
mRNA are maintained so that translational inhibition of SR mimics that of the HAC1
mRNA. Ire1-mediated splicing of this reporter produces a GFP signal that can be quantitatively measured by flow cytometry.
Figure 4. Activation of Ire1(D797N,K799N) continues after WT activity has plateaued. (A) A schematic of the fluorescent splicing reporter (SR) in which the HAC1 ORF was replaced with GFP such that Ire1-mediated splicing of this reporter produces fluorescent GFP. (more ...)
In WT cells, SR fluorescence increased over time with increasing DTT concentration (). At low DTT concentrations (below ~2 mM), GFP levels in WT cells reached a plateau after ~120 min. This plateau, a result of the long half-life of GFP, signifies Ire1 deactivation and is characteristic of an intact homeostatic response that restores the folding capacity of the ER and quells Ire1 signaling.
cells, SR splicing in the first 60–120 min was identical to that observed in WT cells. However, GFP levels continued to rise throughout the time course and its production continued even at doses of DTT to which WT cells adapted (). This phenomenon was most evident when reporter activity was plotted as a function of DTT concentration (). At the 60-min time point, the dose–response curves for both WT and ire1(D797N,K799N)
cells overlapped, indicating that GFP production during the activation phase was equivalent for both WT and mutant enzymes (). In marked contrast, at 240 min the curves deviated substantially (), indicating that after prolonged ER stress Ire1(D797N,K799N) continued to signal at low and intermediate doses of DTT. Note that in these experiments both WT Ire1 and Ire1(D797N,K799N) displayed the same basal activity (; [DTT] = 0.3 mM) and reached the same maximal activity ([DTT] = 3.3 mM), indicating that Ire1 activation by itself was fully intact in the mutant cells (Fig. S3
As a second measure of Ire1 activity, we monitored Ire1 oligomer formation, which can be observed and quantified by fluorescence microscopy as foci in living cells. Oligomer formation closely correlates with HAC1
mRNA splicing and therefore is a powerful tool for monitoring Ire1 activation in vivo (Aragón et al., 2009
). As in our previous studies, we inserted GFP between the transmembrane linker and kinase domains of WT and mutant forms of Ire1, a location that does not interfere with Ire1 function (Aragón et al., 2009
). We measured foci formation of functional WT and mutant Ire1-GFP under conditions at which the adaptation phase dose–response curves of Ire1(D797N,K799N) and WT are most divergent (, [DTT] = 1 mM, dotted line). As shown in , WT Ire1-GFP formed small, transient foci whereas Ire1(D797N,K799N)-GFP formed foci that persisted to the end of the 90-min experiment (). This result is consistent with the observation that WT cells adapted to mild ER stress and shut down Ire1 signaling, while Ire1(D797N,K799N)-GFP activation was sustained in the mutant cells. These data indicate that ire1(D797N,K799N)
cells fail to adapt to prolonged ER stress, suggesting that homeostatic feedback is impaired despite normal induction of UPR target genes.
Ire1(D797N,K799N) cells are able to alleviate ER stress
In principle, the impaired adaptation exhibited in ire1(D797N,K799N)
cells could be due to a failure of the UPR to fix the problem in the ER or to an inability of Ire1 to deactivate once the stress has been relieved. To test the first possibility, we used a reporter of ER redox potential. DTT induces the UPR by shifting the ER redox potential to become more reducing and causes the accumulation of unfolded proteins by blocking disulfide bond formation; UPR induction, in turn, serves to reoxidize the ER lumen. The level of ER stress can be assessed using an ER-targeted redox-sensitive GFP (ero-GFP) reporter (Hanson et al., 2004
; Merksamer et al., 2008
). To test whether ire1(D797N,K799N)
cells restore the oxidizing environment to the ER during sustained UPR insult, cells were treated with 0, 1, or 2 mM DTT () and the ratio of reduced/oxidized ero-GFP (“r/o ratio”) was measured by flow cytometry. In WT cells, the ero-GFP r/o ratio increased upon DTT treatment and then gradually decreased as ero-GFP became reoxidized over the course of the experiment ().
Figure 5. The oxidation potential of the ER is restored in ire1(D797N,K799N) cells. (A–D) Re-oxidation of the ero-GFP reporter occurs in the absence of kinase activity. (A) WT, (B) ire1(D797N,K799N) and (C) ire1Δ cells bearing the ER-targeted redox (more ...)
In ire1(D797N,K799N) cells the basal r/o ratio of ero-GFP was elevated relative to that in WT cells (, 0 mM DTT) and resulted in a relatively smaller fold increase. Despite the diminished dynamic range of the reporter, reoxidation was evident in ire1(D797N,K799N) cells at both concentrations of DTT (), indicating that UPR induction restored the oxidative potential of the ER. By contrast, the ero-GFP r/o ratio in ire1Δ cells showed normal baseline levels and plateaued after DTT addition (). Because these cells are unable to activate the UPR, these data are consistent with the requirement for UPR target gene induction to restore the oxidative environment of the ER.
Deactivation of Ire1(D797N,K799N) is impaired
An unexpected explanation for the elevated baseline of the ero-GFP r/o ratio in ire1(D797N,K799N) cells was provided by observing the intracellular localization of ero-GFP by fluorescence microscopy. In untreated WT cells, ero-GFP was localized to the ER as expected, whereas in ire1(D797N,K799N) cells ero-GFP was partially localized to the cytoplasm both before and after UPR induction (). The cytosolic ero-GFP likely accounts for the higher basal ero-GFP r/o ratio measured in because the cytosol is a reducing environment.
The cytosolic mislocalization of ero-GFP seen in ire1(D797N,K799N) cells was puzzling because our preceding data revealed no differences between WT and ire1(D797N,K799N) cells in the absence of stress. Most relevantly, mislocalization was not observed for Sec63-GFP, which properly localized to the ER in ire1(D797N,K799N) cells (). To confirm that translocation of endogenous ER-targeted proteins was normal, we analyzed the translocation of the ER chaperone Kar2. No difference in Kar2 translocation between untreated WT and ire1(D797N,K799N) cells was observed (unpublished data). We therefore conclude that the high expression levels of the ero-GFP reporter are responsible for its own localization defect in ire1(D797N,K799N) cells. We hypothesize that sustained expression of ER-targeted ero-GFP from a strong constitutive promoter causes chronic ER stress that, in ire1(D797N,K799N) cells, interferes with proper ero-GFP import into the ER.
To understand the nature of this ER translocation impairment, we turned to ire1Δ cells. These cells, which are unable to mount a productive UPR, properly localized ero-GFP to the ER (). The lack of cytosolic ero-GFP signal in ire1Δ cells demonstrates that loss of Ire1 activity is not sufficient to impair ER translocation. Rather, a productive UPR is additionally required to cause the translocation defect observed in ire1(D797N,K799N) cells.
One possibility is that ire1(D797N,K799N) cells fail to adapt to the chronic burden imposed on the ER by ero-GFP expression and do not properly deactivate Ire1. The resulting prolonged UPR signaling would create an overload of ER-targeted proteins, which might overwhelm the capacity of the translocation machinery and cause a back-up of ER client proteins in the cytoplasm. To address this hypothesis, we monitored the abatement of HAC1 mRNA splicing and resolution of Ire1 foci after removal of ER stress. Northern blot analysis revealed that HAC1 mRNA splicing in WT cells declined within 45 min of removing DTT and reset by 90 min after DTT removal (, top). In contrast, ire1(D797N,K799N) cells continued to splice HAC1 mRNA even 120 min after ER stress had been removed (, bottom), indicating that loss of Ire1 kinase activity profoundly delayed Ire1 shut-off. The same trend was observed when we measured Ire1 foci formation: the dissolution of foci in WT cells was noticeable as early as 30 min after DTT washout, whereas Ire1(D797N,K799N) foci were still detectable 120 min after DTT removal (). These data are consistent with the hypothesis that the mechanism of Ire1(D797N,K799N) deactivation is impaired despite the fact that protein folding problems inside the ER are alleviated in these cells.
Figure 6. Shut-off of Ire1(D797N,K799N) is delayed after removal of ER stress. (A) WT or ire1(D797N, K799N) cells were treated with 5 mM DTT for 60 min before DTT washout. Cell samples were taken after DTT washout and total RNA was analyzed by Northern blot for (more ...)
Hyper-phosphorylation of Ire1 is required for rapid de-oligomerization
Mass spectrometry data of the purified cytosolic portion of Ire1 suggest that a 28-amino acid loop (residues 864–892) in the C-terminal end of the kinase domain is highly phosphorylated (unpublished data). We propose that trans-autophosphorylation of this loop (termed HPL for hyper-phosphorylated loop) by Ire1 might contribute to quenching Ire1 activity. If this were true, deletion of HPL in WT IRE1 would mimic the sustained signaling observed in ire1(D797N,K799N) cells, whereas deletion of HPL in Ire1(D797N,K799N) would have no effect on the deactivation phenotype of the mutant protein. To test this possibility, we created ire1ΔHPL-GFP and ire1(D797N,K799N)ΔHPL-GFP cells and monitored attenuation of Ire1 foci after ER stress removal. As shown in , foci in ire1ΔHPL-GFP cells formed readily upon treatment with 5 mM DTT and were sustained substantially longer than in WT control cells after DTT was removed. In contrast, the persistence of foci in ire1(D797N,K799N)ΔHPL-GFP cells mirrored that in ire1(D797N,K799N)-GFP cells (, orange and green lines), indicating that deletion of HPL had no effect on deactivation of Ire1 in the absence of phosphoryl-transfer. Importantly, ire1ΔHPL-GFP cells retained RNase activity as measured by SR splicing (), indicating that activation of Ire1ΔHPL-GFP was intact. Interestingly, the SR splicing phenotype of ire1ΔHPL-GFP cells resembled that of ire1(D797N,K799N) cells, indicating that Ire1ΔHPL has a deactivation defect similar to kinase-inactive Ire1 (compare and ). The kinetics of foci disappearance in ire1ΔHPL cells resembled that in ire1(D797N,K799N) cells (), supporting the hypothesis that the HPL contributes to the regulation of Ire1 shut-off. Notably, however, the phenotype in ire1ΔHPL cells was not as strong as that seen in ire1(D797N,K799N) cells, indicating that phosphorylation of regions outside the HPL must also contribute to Ire1 deactivation.