Mad2 promotes Cyclin B2 recruitment to the kinetochore for guiding accurate mitotic checkpoint.

Accurate mitotic progression relies on the dynamic phosphorylation of multiple substrates by key mitotic kinases. Cyclin-dependent kinase 1 is a master kinase that coordinates mitotic progression and requires its regulatory subunit Cyclin B to ensure full kinase activity and substrate specificity. The function of Cyclin B2, which is a closely related family member of Cyclin B1, remains largely elusive. Here, we show that Mad2 promotes the kinetochore localization of Cyclin B2 and that their interaction at the kinetochores guides accurate chromosome segregation. Our biochemical analyses have characterized the Mad2-Cyclin B2 interaction and delineated a novel Mad2-interacting motif (MIM) on Cyclin B2. The functional importance of the Cyclin B2-Mad2 interaction was demonstrated by real-time imaging in which MIM-deficient mutant Cyclin B2 failed to rescue the chromosomal segregation defects. Taken together, we have delineated a previously undefined function of Cyclin B2 at the kinetochore and have established, in human cells, a mechanism of action by which Mad2 contributes to the spindle checkpoint.

Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis.

Spindle position control is essential for cell fate determination and organogenesis. Early studies indicate the essential role of the evolutionarily conserved Gαi/LGN/NuMA network in spindle positioning. However, the regulatory mechanisms that couple astral microtubules dynamics to the spindle orientation remain elusive. Here we delineated a new mitosis-specific crotonylation-regulated astral microtubule-EB1-NuMA interaction in mitosis. EB1 is a substrate of TIP60, and TIP60-dependent crotonylation of EB1 tunes accurate spindle positioning in mitosis. Mechanistically, TIP60 crotonylation of EB1 at Lys66 forms a dynamic link between accurate attachment of astral microtubules to the lateral cell cortex defined by NuMA-LGN and fine tune of spindle positioning. Real-time imaging of chromosome movements in HeLa cells expressing genetically encoded crotonylated EB1 revealed the importance of crotonylation dynamics for accurate control of spindle orientation during metaphase-anaphase transition. These findings delineate a general signaling cascade that integrates protein crotonylation with accurate spindle positioning for chromosome stability in mitosis.

Phase separation drives decision making in cell division.

Liquid-liquid phase separation (LLPS) of biomolecules drives the formation of subcellular compartments with distinct physicochemical properties. These compartments, free of lipid bilayers and therefore called membraneless organelles, include nucleoli, centrosomes, heterochromatin, and centromeres. These have emerged as a new paradigm to account for subcellular organization and cell fate decisions. Here we summarize recent studies linking LLPS to mitotic spindle, heterochromatin, and centromere assembly and their plasticity controls in the context of the cell division cycle, highlighting a functional role for phase behavior and material properties of proteins assembled onto heterochromatin, centromeres, and central spindles via LLPS. The techniques and tools for visualizing and harnessing membraneless organelle dynamics and plasticity in mitosis are also discussed, as is the potential for these discoveries to promote new research directions for investigating chromosome dynamics, plasticity, and interchromosome interactions in the decision-making process during mitosis.

Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation.

Faithful segregation of chromosomes in mammalian cells requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation, which triggers mitotic entry, have been extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via the acetyltransferase TIP60 in human cell division. CDK1-cyclin B phosphorylates Ser90 of TIP60, which elicits TIP60-dependent acetylation of Aurora B and promotes accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protects Aurora B's activation loop from dephosphorylation by the phosphatase PP2A to ensure a robust, error-free metaphase-anaphase transition. These findings delineate a conserved signaling cascade that integrates protein phosphorylation and acetylation with cell cycle progression for maintenance of genomic stability.

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment.

The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C-MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.

The Microtubule Plus End Tracking Protein TIP150 Interacts with Cortactin to Steer Directional Cell Migration.

Cell migration is orchestrated by dynamic interactions of microtubules with the plasma membrane cortex. How these interactions facilitate these dynamic processes is still being actively investigated. TIP150 is a newly characterized microtubule plus end tracking protein essential for mitosis and entosis (Ward, T., Wang, M., Liu, X., Wang, Z., Xia, P., Chu, Y., Wang, X., Liu, L., Jiang, K., Yu, H., Yan, M., Wang, J., Hill, D. L., Huang, Y., Zhu, T., and Yao, X. (2013) Regulation of a dynamic interaction between two microtubule-binding proteins, EB1 and TIP150, by the mitotic p300/CBP-associated factor (PCAF) orchestrates kinetochore microtubule plasticity and chromosome stability during mitosis. J. Biol. Chem. 288, 15771-15785; Xia, P., Zhou, J., Song, X., Wu, B., Liu, X., Li, D., Zhang, S., Wang, Z., Yu, H., Ward, T., Zhang, J., Li, Y., Wang, X., Chen, Y., Guo, Z., and Yao, X. (2014) Aurora A orchestrates entosis by regulating a dynamic MCAK-TIP150 interaction. J. Mol. Cell Biol. 6, 240-254). Here we show that TIP150 links dynamic microtubules to steer cell migration by interacting with cortactin. Mechanistically, TIP150 binds to cortactin via its C-terminal tail. Interestingly, the C-terminal TIP150 proline-rich region (CT150) binds to the Src homology 3 domain of cortactin specifically, and such an interaction is negatively regulated by EGF-elicited tyrosine phosphorylation of cortactin. Importantly, suppression of TIP150 or overexpression of phospho-mimicking cortactin inhibits polarized cell migration. In addition, CT150 disrupts the biochemical interaction between TIP150 and cortactin in vitro, and perturbation of the TIP150-cortactin interaction in vivo using a membrane-permeable TAT-CT150 peptide results in an inhibition of directional cell migration. We reason that a dynamic TIP150-cortactin interaction orchestrates directional cell migration via coupling dynamic microtubule plus ends to the cortical cytoskeleton.

Signaling Scaffold Protein IQGAP1 Interacts with Microtubule Plus-end Tracking Protein SKAP and Links Dynamic Microtubule Plus-end to Steer Cell Migration.

Cell migration is orchestrated by dynamic interaction of microtubules with the plasma membrane cortex. However, the regulatory mechanisms underlying the cortical actin cytoskeleton and microtubule dynamics are less characterized. Our earlier study showed that small GTPase-activating proteins, IQGAPs, regulate polarized secretion in epithelial cells (1). Here, we show that IQGAP1 links dynamic microtubules to steer cell migration via interacting with the plus-end tracking protein, SKAP. Biochemical characterizations revealed that IQGAP1 and SKAP form a cognate complex and that their binding interfaces map to the WWIQ motif and the C-terminal of SKAP, respectively. The WWIQ peptide disrupts the biochemical interaction between IQGAP1 and SKAP in vitro, and perturbation of the IQGAP1-SKAP interaction in vivo using a membrane-permeable TAT-WWIQ peptide results in inhibition of directional cell migration elicited by EGF. Mechanistically, the N-terminal of SKAP binds to EB1, and its C terminus binds to IQGAP1 in migrating cells. Thus, we reason that a novel IQGAP1 complex orchestrates directional cell migration via coupling dynamic microtubule plus-ends to the cell cortex.

Regulation of a dynamic interaction between two microtubule-binding proteins, EB1 and TIP150, by the mitotic p300/CBP-associated factor (PCAF) orchestrates kinetochore microtubule plasticity and chromosome stability during mitosis.

The microtubule cytoskeleton network orchestrates cellular dynamics and chromosome stability in mitosis. Although tubulin acetylation is essential for cellular plasticity, it has remained elusive how kinetochore microtubule plus-end dynamics are regulated by p300/CBP-associated factor (PCAF) acetylation in mitosis. Here, we demonstrate that the plus-end tracking protein, TIP150, regulates dynamic kinetochore-microtubule attachments by promoting the stability of spindle microtubule plus-ends. Suppression of TIP150 by siRNA results in metaphase alignment delays and perturbations in chromosome biorientation. TIP150 is a tetramer that binds an end-binding protein (EB1) dimer through the C-terminal domains, and overexpression of the C-terminal TIP150 or disruption of the TIP150-EB1 interface by a membrane-permeable peptide perturbs chromosome segregation. Acetylation of EB1-PCAF regulates the TIP150 interaction, and persistent acetylation perturbs EB1-TIP150 interaction and accurate metaphase alignment, resulting in spindle checkpoint activation. Suppression of the mitotic checkpoint serine/threonine protein kinase, BubR1, overrides mitotic arrest induced by impaired EB1-TIP150 interaction, but cells exhibit whole chromosome aneuploidy. Thus, the results identify a mechanism by which the TIP150-EB1 interaction governs kinetochore microtubule plus-end plasticity and establish that the temporal control of the TIP150-EB1 interaction by PCAF acetylation ensures chromosome stability in mitosis.

CSPP1 stabilizes microtubules by capping both plus and minus ends.

Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting polymerization. Importantly, CSPP1-bound MTs were resistant to mitotic centromere-associated kinesin-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

Phase separation of EB1 guides microtubule plus-end dynamics.

In eukaryotes, end-binding (EB) proteins serve as a hub for orchestrating microtubule dynamics and are essential for cellular dynamics and organelle movements. EB proteins modulate structural transitions at growing microtubule ends by recognizing and promoting an intermediate state generated during GTP hydrolysis. However, the molecular mechanisms and physiochemical properties of the EB1 interaction network remain elusive. Here we show that EB1 formed molecular condensates through liquid-liquid phase separation (LLPS) to constitute the microtubule plus-end machinery. EB1 LLPS is driven by multivalent interactions among different segments, which are modulated by charged residues in the linker region. Phase-separated EB1 provided a compartment for enriching tubulin dimers and other plus-end tracking proteins. Real-time imaging of chromosome segregation in HeLa cells expressing LLPS-deficient EB1 mutants revealed the importance of EB1 LLPS dynamics in mitotic chromosome movements. These findings demonstrate that EB1 forms a distinct physical and biochemical membraneless-organelle via multivalent interactions that guide microtubule dynamics.

Structural insights into human CCAN complex assembled onto DNA.

In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.

Curcumin, a dietary component, has anticancer, chemosensitization, and radiosensitization effects by down-regulating the MDM2 oncogene through the PI3K/mTOR/ETS2 pathway.

The oncoprotein MDM2, a major ubiquitin E3 ligase of tumor suppressor p53, has been suggested as a novel target for human cancer therapy based on its p53-dependent and p53-independent activities. We have identified curcumin, which has previously been shown to have anticancer activity, as an inhibitor of MDM2 expression. Curcumin down-regulates MDM2, independent of p53. In a human prostate cancer cell lines PC3 (p53(null)), curcumin reduced MDM2 protein and mRNA in a dose- and time-dependent manner, and enhanced the expression of the tumor suppressor p21(Waf1/CIP1). The inhibitory effects occur at the transcriptional level and seem to involve the phosphatidylinositol 3-kinase/mammalian target of rapamycin/erythroblastosis virus transcription factor 2 pathway. Curcumin induced apoptosis and inhibited proliferation of PC3 cells in culture, but both MDM2 overexpression and knockdown reduced these effects. Curcumin also inhibited the growth of these cells and enhanced the cytotoxic effects of gemcitabine. When it was administered to tumor-bearing nude mice, curcumin inhibited growth of PC3 xenografts and enhanced the antitumor effects of gemcitabine and radiation. In these tumors, curcumin reduced the expression of MDM2. Down-regulation of the MDM2 oncogene by curcumin is a novel mechanism of action that may be essential for its chemopreventive and chemotherapeutic effects. Our observations help to elucidate the process by which mitogens up-regulate MDM2, independent of p53, and identify a mechanism by which curcumin functions as an anticancer agent.

Dexamethasone as a chemosensitizer for breast cancer chemotherapy: potentiation of the antitumor activity of adriamycin, modulation of cytokine expression, and pharmacokinetics.

Dexamethasone (DEX) is mainly used as an anti-emetic agent in cancer therapy. We have recently demonstrated that DEX pretreatment increases the antitumor activity of the cancer chemotherapeutic agents carboplatin and gemcitabine, and decreases host toxicity in nude mouse xenograft models of human cancer. However, the underlying mechanisms are not fully understood. The present study was designed to determine the effects of DEX pretreatment on the anticancer activity of adriamycin (ADR) in a syngeneic model of breast cancer (4T1), emphasizing the effects of DEX on cytokine expression and modulation of ADR pharmacokinetics. We have demonstrated five major new findings about DEX pretreatment: a) it enhances the therapeutic effect of ADR, inducing almost complete inhibition of tumor growth; b) it increases tumor ADR accumulation; c) it modulates the expression of cytokines produced by the tumor, increasing TNFalpha and decreasing IL-1beta and VEGF expression; d) it enhances the effects of ADR on induction of apoptosis and inhibition of cell proliferation; and e) it suppresses nuclear NFkappaB activation and inhibits ADR-induced NFkappaB activation, possibly via IkappaB up-regulation. These findings suggest that DEX can be used as a chemosensitizer and chemoprotectant. These results provide a rationale for the expanded clinical use of DEX for cancer therapy.

In vitro anti-cancer activity and structure-activity relationships of natural products isolated from fruits of Panax ginseng.

PURPOSE: Panax ginseng and its extracts have long been used for medical purposes; there is increasing interest in developing ginseng products as cancer preventive or therapeutic agents. The present study was designed to determine biological structure-activity relationships (SAR) for saponins present in Panax ginseng fruits. METHODS: Eleven saponins were extracted from P. ginseng fruits and purified by use of D(101) resin and ordinary and reverse-phase silica gel column chromatography. Their chemical structures were elucidated on the basis of physicochemical constants and NMR spectra. Compounds were then evaluated for SAR with their in vitro cytotoxicity against several human cancer cell lines. RESULTS: The 11 compounds were identified as 20(R)-dammarane-3beta,12beta,20,25-tetrol (25-OH-PPD, 1); 20(R)-dammarane-3beta,6alpha,12beta,20,25-pentol (25-OH-PPT, 2); 20(S)-protopanaxadiol (PPD, 3); daucosterine 4, 20(S)-ginsenoside-Rh(2) (Rh(2), 5); 20(S)-ginsenoside-Rg(3) (Rg(3,) 6); 20(S)-ginsenoside-Rg(2) (Rg(2), 7); 20(S)-ginsenoside-Rg(1) (Rg(1), 8); 20(S)-ginsenoside-Rd (Rd, 9); 20(S)-ginsenoside-Re (Re, 10); and 20(S)-ginsenoside-Rb(1) (Rb(1), 11). Among the eleven compounds, 1, 3 and 5 were the most effective inhibitors of cell growth and proliferation and inducers of apoptosis and cell cycle arrest. For 1, the IC(50) values for most cell lines were in the range of 10-60 microM, at least twofold lower than for any of the other compounds. Compounds 1 and 3 had significant, dose-dependent effects on apoptosis, proliferation, and cell cycle progression. CONCLUSIONS: The results suggest that the type of dammarane, the number of sugar moieties, and differences in the substituent groups affect their anti-cancer activity. This information may be useful for evaluating the structure/function relationship of other ginsenosides and their aglycones and for development of novel anticancer agents.

Genistein, a dietary isoflavone, down-regulates the MDM2 oncogene at both transcriptional and posttranslational levels.

Although genistein has chemopreventive effects in several human malignancies, including cancers of the breast, colon, and prostate, the mechanisms of action are not fully understood. Herein we report novel mechanisms whereby genistein down-regulates the MDM2 oncogene, perhaps explaining some of its anticancer activities. In a dose- and time-dependent manner, genistein reduced MDM2 protein and mRNA levels in human cell lines of breast, colon, and prostate cancer; primary fibroblasts; and breast epithelial cells. The inhibitory effects were found at both transcriptional and posttranslational levels and were independent of tyrosine kinase pathways. We found that the NFAT transcription site in the region between -132 and +33 in the MDM2 P2 promoter was responsive to genistein. At the posttranslational level, genistein induced ubiquitination of MDM2, which led to its degradation. Additionally, genistein induced apoptosis and G2 arrest and inhibited proliferation in a variety of human cancer cell lines, regardless of p53 status. We further showed that MDM2 overexpression abrogated genistein-induced apoptosis in vitro and that genistein inhibited MDM2 expression and tumor growth in PC3 xenografts. In conclusion, genistein directly down-regulates the MDM2 oncogene, representing a novel mechanism of its action that may have implications for its chemopreventive and chemotherapeutic effects.

Preclinical pharmacology of the novel antitumor agent adaphostin, a tyrphostin analog that inhibits bcr/abl.

PURPOSE: To define several pharmacological properties for the potential anticancer agent, adaphostin, in order to determine whether the compound is appropriate for clinical evaluation as an anticancer agent. METHODS: The analytical procedure involved high-performance liquid chromatography and utilized an analytical J'Sphere ODS H-80 column. RESULTS: The stability of adaphostin at two different concentrations was determined at temperatures of 37 degrees C, 4 degrees C, and -80 degrees C, in the plasma of mice, rats, dogs, and humans. The compound was most stable at the lower temperatures. At all temperatures, adaphostin was generally most stable in human plasma and least stable in dog plasma. Adaphostin bound strongly (>93%) to proteins in plasma from all four species. Following intravenous (i.v.) administration to mice (50 mg/kg; 150 mg/m(2)), plasma concentrations declined rapidly from 50 microM at 2 min to 1 microM at 2 h. Elimination was triexponential, with t (1/2) values of 1.1, 9.1, and 41.2 min. The Cl(tb) was 0.411 L/(min.m(2)), the V (dss) was 24.6 L/m(2), and the AUC was 927 microM.min. In a comparison of vehicles for intraperitoneal (i.p.) dosing, PEG 300 allowed the highest plasma concentrations of adaphostin. Bioavailability following an i.p. dose was greater than that following a subcutaneous dose, or that for a dose administered by oral gavage. For rats dosed i.v. with adaphostin (50 mg/kg; 300 mg/m(2)), plasma concentrations also decreased triexponentially, with t (1/2) values of 1.8, 10.6, and 136 min. Other pharmacokinetic values were Cl(tb) = 0.466 L/(min.m(2)), AUC = 1,161 microM.min, and V (dss)=8.0 L/m(2). Analysis of samples collected from two dogs dosed i.v. with adaphostin (7.5 mg/kg; 150 mg/m(2)) showed that plasma concentrations decreased in a biphasic manner, with individual values for t (1/2alpha) of 6.0 and 9.8 min for the distribution phase and t (1/2beta) of 40.6 and 66.2 min for the elimination phase. Other pharmacokinetic values were Cl(tb) = 0.565 and 0.852 L/(min.m(2)), AUC = 673 and 446 microM min, and V (dss) = 29.6 and 56.8 L/m(2). CONCLUSIONS: The stability of adaphostin in plasma varies with species. In mice and dogs dosed with adaphostin, plasma concentrations of the compound decreased rapidly. The clearance of adaphostin from plasma, on an m(2) basis, was equivalent for mice and rats but more rapid in dogs. These results are relevant for assessing the pharmacologic and toxicologic profiles and the antitumor activity of adaphostin in humans.

Determination of desoxyepothilone B in nude mice plasma by liquid-liquid extraction and reversed-phase high-performance liquid chromatography.

A novel reversed-phase high-performance liquid chromatographic (HPLC) method has been established for the determination of a newly developed anti-cancer agent desoxyepothilone B (dEpo B) in nude mice plasma. The sample preparation involved deproteination of 200 microl of plasma sample first, followed by liquid-liquid extraction of the resultant supernatant with chloroform. The compound taxol was used as the internal standard. Chromatographic separations were carried out on a 250 mm x 4.6 mm Zorbax SB-phenyl column with acetonitrile-0.25% orthophosphoric acid (50/50, v/v) as mobile phase and UV detection at 250 nm. For dEpo B and taxol at the concentration level of 10 microg/ml in nude mice plasma, the absolute extraction recoveries were 85.3 and 87.2%, respectively. The linear quantification range of the method was 0.1-100 microg/ml in nude mice plasma with linear correlation coefficients greater than 0.999. The within-day and between-day relative standard deviations (R.S.D.s) for dEpo B at 0.5, 2.5 and 10 microg/ml levels in nude mice plasma fell in the range of 2.8-4.8 and 1.5-4.6%, and the within-day and between-day recoveries were in the range of 96.5-101.7 and 97.7-101.2%, respectively.

IRE1-RACK1 axis orchestrates ER stress preconditioning-elicited cytoprotection from ischemia/reperfusion injury in liver.

Endoplasmic reticulum (ER) stress is involved in ischemic preconditioning that protects various organs from ischemia/reperfusion (I/R) injury. We established an in vivo ER stress preconditioning model in which tunicamycin was injected into rats before hepatic I/R. The hepatic I/R injury, demonstrated by serum aminotransferase level and the ultra-structure of the liver, was alleviated by administration of tunicamycin, which induced ER stress in rat liver by activating inositol-requiring enzyme 1 (IRE1) and upregulating 78 kDa glucose-regulated protein (GRP78). The proteomic identification for IRE1 binders revealed interaction and cooperation among receptor for activated C kinase 1 (RACK1), phosphorylated AMPK, and IRE1 under ER stress conditions in a spatiotemporal manner. Furthermore, in vitro ER stress preconditioning was induced by thapsigargin and tunicamycin in L02 and HepG2 cells. Surprisingly, BCL2 was found to be phosphorylated by IRE1 under ER stress conditions to prevent apoptotic process by activation of autophagy. In conclusion, ER stress preconditioning protects against hepatic I/R injury, which is orchestrated by IRE1-RACK1 axis through the activation of BCL2. Our findings provide novel insights into the molecular pathways underlying ER stress preconditioning-elicited cytoprotective effect against hepatic I/R injury.