An AKT1- and TRIM21-mediated Phosphodegron Controls Proteasomal Degradation of HuR Enabling Cell Survival under Heat Shock

doi:10.21203/rs.3.rs-1774410/v1

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An AKT1- and TRIM21-mediated Phosphodegron Controls Proteasomal Degradation of HuR Enabling Cell Survival under Heat Shock

https://doi.org/10.21203/rs.3.rs-1774410/v1

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posted 23 Jun, 2022

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Post-transcriptional regulation by RNA-binding proteins (RBPs) is a major mode of controlling gene expression under stress conditions. The RBP HuR regulates the translation and/or stability of mRNAs of multiple genes involved in stress responses. HuR is degraded in response to heat stress consequent to ubiquitination of the K182 amino acid residue. We have identified TRIM21 as the E3-ubiquitin ligase causing HuR polyubiquitination at K182 and proteasomal degradation under heat shock. TRIM21-mediated degradation of HuR protected cells from heat-shock induced cell death. By a combination of molecular dynamics simulation and experimentation we found the S100 and E101 residues to be required for binding of TRIM21 to HuR. Heat shock-induced phosphorylation of S100 was necessary for TRIM21 interaction with HuR and subsequent degradation. We identified AKT1 as the kinase which phosphorylates S100, allowing the recognition of HuR by TRIM21. Sequential phosphorylation by AKT1 and ubiquitination by TRIM21 therefore determine a “phosphodegron” in HuR that regulates the cellular level of HuR under heat shock, thereby enabling a crucial adaptive mechanism allowing cell survival in response to heat stress.

Cellular stress activates multiple decision-making responses in gene expression pathways allowing cells to survive and adapt to stress conditions or undergo cell death¹. Cellular stress responses therefore involve stress-specific transcriptional and post-transcriptional changes in gene expression^2,3. Post-transcriptional regulation of gene expression is mediated by RNA-binding proteins (RBPs) and non-coding RNAs (ncRNAs) which regulate the translation and turnover of mRNAs^4–6. Stress-induced regulation of RBP abundance and activity is therefore a widespread phenomenon of central importance in stress responses⁷.

Heat shock response (HSR) is an evolutionarily conserved, fundamental stress response in organisms to cope with sudden changes in ambient temperature. Heat shock leads to deleterious effects on the cellular cytoskeleton together with unfolding of proteins, changes in cellular metabolism and gene expression, leading to cell cycle arrest⁸. Depending on duration and severity, heat stress can result in cell death⁹. Transcriptional and post-transcriptional changes in gene expression in heat-stressed cells lead to the altered expression of molecular chaperones and other regulators which enable the cells to cope with the stress^10–13. As heat stress induces a global inhibition of translation, specific RBPs are regulated to ensure the continued stability and translation of a cohort of mRNAs required for the HSR^14–16. Regulation of RBP levels and function is therefore crucial for HSR.

HuR or ELAVL1 is a ubiquitously expressed RBP involved in the cellular response to a variety of stress conditions^17–22. HuR determines the cellular stress responses by regulating the stability and/or translation of mRNAs of multiple stress-responsive genes²³. HuR is predominantly nuclear but translocates to the cytoplasm in response to most stress stimuli, where it exerts its post-transcriptional effects on target mRNAs²⁴. HuR undergoes partial nuclear-cytoplasmic translocation in response to heat shock at 45°C and localize in cytoplasmic foci²⁵. Conversely, the majority of HuR was found to crosslink with nuclear poly(A) RNA in response to heat shock, suggesting that HuR gets trapped with nuclear mRNAs in heat-stressed cells ²². Interestingly, mild heat shock at 43°C strongly reduced cellular HuR level by promoting the degradation of HuR through ubiquitination of the Lys182 (K182) residue²⁶. Recently, K182 was also found to be the target of ubiquitination in HuR in response to UV irradiation²⁷. However, neither the E3 ubiquitin ligase responsible for this heat shock-induced proteolysis of HuR, nor the specific post-translational modifications leading to the recognition by the E3 ligase have been identified.

In this study we have identified TRIM21 as the E3-ubiquitin ligase causing HuR poly-ubiquitination at K182 and proteasomal degradation under heat shock. The residues S100 and E101 were found to be crucial for the recognition of HuR by TRIM21. Remarkably, phosphorylation of S100 by the kinase AKT1 was required for the interaction with TRIM21. The AKT1 and TRIM21-mediated phosphorylation and degradation of HuR protected cells from heat-shock induced death. AKT1 and TRIM21 therefore determine a “phosphodegron” in HuR that is crucial for the degradation of HuR and the cellular response to thermal stress.

The E3 ubiquitin ligase TRIM21 causes degradation of HuR in response to heat shock

Moderate heat shock, at 43°C for 2 h, to MCF7 cells in 10% serum-containing medium caused a strong time-dependent reduction of HuR protein to ~25% of initial level, with no change in mRNA level (Fig. 1A). Heat shock at 43°C to MCF7 cells in serum-free medium also caused a similar reduction in HuR protein level, as reported earlier in HeLa cells²⁶, suggesting that the HuR response to heat shock is independent of serum or cell type (Suppl. Fig. 1). Exogenously expressed EGFP-tagged HuR showed a similar reduction in response to heat shock (Fig. 1B). Both nuclear and cytoplasmic HuR showed similar degradation in heat-stressed cells, indicating cytoplasmic translocation as not a prerequisite for HuR degradation (Suppl. Fig. 2). Protein synthesis inhibition by cycloheximide (CHX) rapidly reduced HuR in heat-stressed cells compared to cells at 37 °C, demonstrating a significantly higher degradation rate of HuR under heat shock. However, treatment with the proteasomal inhibitor MG132, in presence of CHX, maintained the cellular level of HuR at 43°C (Fig. 1C). The half-life of HuR in heat-stressed cells treated with CHX goes down to ~30 min, whereas it is restored to more than 2 h on MG132 treatment (Fig 1D). This demonstrates that HuR is proteasomally degraded under heat shock.

As the K182 residue of HuR is reported to be polyubiquitinated under heat shock, we expressed 6X-His-tagged wild type (WT) and K182R mutant HuR proteins in cells and subjected them to heat shock. WT HuR was efficiently degraded, whereas K182R mutant HuR was protected from degradation under heat shock (Fig 1E). WT HuR underwent polyubiquitination in cells exposed to heat shock which was significantly reduced in case of the K182R mutant (Fig. 1F).

The K182 residue is polyubiquitinated by the ubiquitin ligase TRIM21 in response to UVC irradiation²⁷. We therefore investigated whether TRIM21 was the E3-ligase responsible for degrading HuR under heat shock. HuR and TRIM21 showed interaction in cells at normal temperature and enhanced interaction in cells exposed to heat shock at 43°C for 1 h and treated with MG132 (Fig. 1G). siRNA-mediated depletion of TRIM21 prevented the degradation whereas overexpression of TRIM21 enhanced the degradation of HuR under heat shock (Fig. 1H and 1I).

To determine the involvement of K182 in the degradation of HuR by TRIM21 under heat shock, TRIM21 was co-expressed in cells expressing either myc-tagged WT or K182R mutant HuR. Upon heat shock, WT-HuR was degraded but the HuR K182R mutant was protected from degradation (Fig. 1J). K182R mutant HuR also showed significantly reduced interaction with TRIM21 upon heat shock (Fig. 1K). Together, these observations demonstrated TRIM21 as the E3 ubiquitin ligase causing the degradation of HuR under heat shock via the ubiquitination of K182.

TRIM21-mediated degradation of HuR protects cells from heat shock-induced cell death

TRIM21 enhances cell proliferation and reverses the DNA damage-induced cell death in UV-irradiated cells²⁸. We therefore investigated the effect of TRIM21 on cell viability and death in response to heat shock. TRIM21 overexpression enhanced cell viability at 37°C and also significantly rescued the viability of heat-stressed cells (Fig. 2A, left panel). Conversely, TRIM21 depletion reduced cell viability and further accentuated the reduction of viability of heat-stressed cells (Fig. 2A, right panel). TRIM21 overexpression reduced caspase 3/7 activity in cells at 37°C and also significantly reduced the enhanced caspase activity in heat-stressed cells (Fig. 2B, left panel). The opposite effect was seen on TRIM21 knockdown (Fig. 2B, right panel). Annexin V/PI staining of cells overexpressing TRIM21 showed significant reduction in the enhanced apoptosis of cells exposed to heat shock (Fig. 2C). The converse was observed in cells in which TRIM21 was knocked down (Fig. 2D). This demonstrated that TRIM21 enhanced the viability and reduced apoptosis of heat- stressed cells.

We then investigated the effect of TRIM21 on the morphological changes of heat-stressed cells. Heat shock caused a visible change in cellular morphology in comparison to cells at 37°C (Suppl. Fig. 3 and 4). The reduction in cell area and perimeter upon heat shock were significantly restored on TRIM21 overexpression (Fig. 2E). Conversely, TRIM21 knockdown further reduced cell area and perimeter of heat stressed cells (Fig. 2F). TRIM21 also enhanced the long-term viability and proliferation of heat-stressed cells. TRIM21 overexpression significantly enhanced the proliferation of cells at 37°C over a 72 h period. While cells exposed to heat shock showed significantly reduced proliferation during this period, TRIM21 overexpression significantly enhanced the proliferation of heat-stressed cells and restored it nearly to the level of cells unexposed to heat shock (Fig. 2G). Depletion of TRIM21 showed the opposite effect (Fig. 2H). Heat-stressed cells formed significantly less number of colonies compared to cells not exposed to heat shock, whereas colony formation by heat-stressed cells overexpressing TRIM21 was nearly similar (Fig. 2I). Together, these observations demonstrated a profound role of TRIM21 in ensuring short term survival and long term viability of cells exposed to heat shock.

We thereafter investigated whether the survival advantage provided by TRIM21 under heat shock was mediated by its degradation of HuR. Overexpression of both WT HuR and HuR K182R, which is refractory to TRIM21-mediated degradation, significantly reduced the enhanced viability of cells observed on TRIM21 overexpression. WT HuR reduced the enhanced viability of heat-stressed cells overexpressing TRIM21 which was further reduced on overexpression of HuR K182R mutant (Fig. 2J, left panel). The opposite effect was observed in case of cellular caspase activity (Fig. 2J, right panel). This demonstrated that WT HuR, and more so the non-degradable K182R mutant HuR, could reverse the increase in cell viability and decrease in apoptosis induced by TRIM21 overexpression in heat-stressed cells. WT HuR and K182R mutant HuR also reduced the enhancement in the proliferation rate caused by TRIM21 overexpression in cells exposed to heat shock (Fig. 2K). Together, these findings demonstrated that TRIM21’s ability to protect cells from heat shock-induced death is, at least partly, mediated by its degradation of HuR.

S100 and E101 residues of HuR are determinants of recognition by TRIM21

Heat shock induced the interaction between HuR and TRIM21 suggesting the presence of specific determinants of TRIM21 binding in HuR which might be post-translationally modified in response to heat shock. We adopted an unbiased approach to delineate the determinants of HuR interaction with TRIM21 under heat shock. His-tagged versions of various deletion mutants, consisting of different domains of the HuR protein, were constructed (Fig. 3A). When expressed in cells exposed to heat shock, only WT HuR and RRM1-2 showed degradation, indicating that the region of HuR interacting with TRIM21 was localized in RRM1-2 (Fig. 3B). Interestingly, RRM2-3, which still contained the K182 residue, did not show degradation under heat shock, suggesting that residues necessary for substrate recognition by TRIM2 were present in RRM1 or in the linker region. Also, only WT HuR and HuR RRM1-2 interacted with TRIM21, further indicating that RRM1-2 contained the residues crucial for recognition of HuR by TRIM21 (Fig. 3C).

We then adopted molecular dynamics (MD) simulation to identify the putative contact points between HuR and TRIM21. The MD simulation was performed with HuR RRM1-2 (PDB: 4EGL) and the PRYSPRY domain of TRIM21 (PDB: 2IWG), that has been shown to be responsible for substrate binding²⁹. The simulation showed a rapid reduction and subsequent stabilization of the solvent accessible surface area (SASA) of the system indicating interaction between the two molecules (Suppl. Fig. 5A). The contact area was around 30 nm² and remained nearly constant throughout the length of the simulation post binding. The interaction was also energetically stable, being of the order of 10³ kJ/mol and was mostly electrostatic (Suppl. Fig. 5B). The putative contact surface between HuR and TRIM21 showed two regions of HuR, between 98-106 amino acids and between 111-119 amino acids. Among these residues, the highest probability of contact was obtained for two amino acids, S100 and E101, located in the linker region between RRMs 1 and 2 (Fig. 3D). Another two amino acids, R115 and T116, also showed interaction with TRIM21 residues but with lower probability. The molecular distance between S100 and K182, the site of TRIM21-mediated ubiquitination, was estimated to be 24.19 Å, whereas the distance between R115 and K182 was 19.22 Å (Fig. 3E). However, K182 and S100 are located on the same face of HuR, whereas R115 is located on the opposite face. The MD simulation results allowed the molecular docking of HuR RRM1-2 and TRIM21 PRYSPRY domains, showing a close contact between S100 residue of HuR and TRIM21 (Fig. 3F). The MD simulation and molecular docking results suggested S100 and E101 residues as the possible primary determinants of interaction between HuR and TRIM21.

S100 and E101 residues of HuR are required for TRIM21 binding and degradation of HuR under heat shock

The S100 and E101 residues of HuR were mutated to generate HuR S100A and E101A mutants and a S100A/E101A double mutant (Fig. 4A). All the three mutant HuR proteins were found to be refractory to heat shock-induced degradation while WT HuR was degraded efficiently (Fig. 4B). TRIM21 failed to interact with either of the single mutants or the double mutant, while it interacted efficiently with WT HuR (Fig. 4C). The S100A/E101A double mutant HuR also showed reduced ubiquitination compared to WT HuR under heat shock, (Fig. 4D). This demonstrated S100 and E101 residues as necessary for the heat shock-induced ubiquitination and degradation of HuR by TRIM21.

We checked the effect of the HuR S100A/E101A mutant on cell viability and apoptosis after exposure to heat shock. Overexpression of the HuR S100A/E101A double mutant significantly reversed the enhancement of cell viability and reduction in caspase activity observed upon TRIM21 overexpression both in cells exposed to 37°C and 43°C (Fig. 4E, left top and bottom panels). Similarly, overexpression of the HuR S100A/E101A mutant reversed the reduction in apoptosis of heat-stressed cells upon TRIM21 overexpression (Fig. 4E, right top and bottom panels and Suppl. Fig 6). The HuR double mutant also significantly reduced the enhanced proliferation of cells overexpressing TRIM21 during the 72 h period post exposure to heat shock (Fig. 4F). Therefore, the degradation-resistant S100A/E101A double mutant HuR could counteract TRIM21’s ability to ensure cell survival and proliferation under heat shock.

We also tested whether the R115 and T116 residues were required for degradation of HuR and interaction with TRIM21. A R115A/T116A double mutant HuR showed degradation similar to WT HuR upon exposure to heat shock (Suppl. Fig. 7A). Also the R115A/T116A double mutant showed interaction with TRIM21 in cells exposed to heat shock (Suppl. Fig. 7B). Therefore, the R115 and T116A residues did not determine the interaction between HuR and TRIM21 and did not contribute to HuR degradation under heat shock.

Finally, we determined the effect of the S100A/E101A mutant on the mRNA levels of some of the post-transcriptional targets of HuR which are related to cell proliferation, apoptosis and stress responses. qRT-PCR for specific HuR target mRNAs showed that these mRNA levels were strongly reduced in WT HuR-overexpressing cells exposed to 43 °C, whereas overexpression of HuR S100A/E101A double mutant restored these mRNAs to nearly normal levels (Fig. 4G). Also, HuR S100A/E100A double mutant retained the ability to interact with these mRNAs (Suppl. Fig. 8). This indicated that the degradation resistant HuR double mutant protein could bind with and stabilize the HuR target mRNAs which are otherwise destabilized in heat-stressed cells.

Phosphorylation of S100 is required for TRIM21 binding and degradation of HuR under heat shock

As phosphorylation of HuR have been shown to influence its sub-cellular localization and mRNA-binding ability under different stress conditions^24,30, we investigated whether S100 was phosphorylated in response to heat shock. WT HuR showed high level of serine phosphorylation in heat stressed cells whereas the S100A mutant showed strongly reduced serine phosphorylation, demonstrating S100 as a target for phosphorylation under heat shock (Fig. 5A). A phosphomimetic S100D mutant was degraded in cells even at 37°C (Fig. 5B). However, knockdown of TRIM21 prevented the degradation of HuR S100D mutant in the absence of heat shock (Fig. 5C). TRIM21 also efficiently interacted with the HuR S100D mutant both in absence and presence of heat shock, whereas it interacted efficiently with WT HuR only upon exposure to heat shock (Fig. 5D). Together, these data indicated that the phosphorylation of S100 was responsible for recognition of HuR by TRIM21 and subsequent degradation. We also investigated the effect of the S100D mutant on cell viability and apoptosis in response to heat shock. Overexpression of WT HuR reduced cell viability and enhanced caspase activity in heat-stressed cells, as observed before, whereas overexpression of the S100D mutant showed the opposite effect (Fig. 5 E and F). Therefore, the rapid degradation of the HuR S100D mutant provided the same survival advantage to the cells caused by TRIM21-mediated degradation of HuR under heat shock.

AKT1 phosphorylates S100 leading to TRIM21-mediated degradation of HuR under heat shock

We then proceeded to identify the kinase phosphorylating S100 of HuR in response to heat shock. S100 is part of a kinase substrate motif RXXS/T (R97PSS100) which was found to be conserved across vertebrates (Fig. 6A). One of the kinases with the substrate motif RXXS/T is AKT1, which plays a central role in mediating critical cellular responses including cell growth and survival^31,32. AKT1 is also reported to be induced by heat shock and involved in suppression of heat shock-induced cell death^33,34. Therefore we tested whether AKT1 might be the kinase phosphorylating HuR in response to heat shock. Treatment of cells with an Akt inhibitor prevented the degradation of HuR in heat stressed cells (Fig. 6B). siRNA-mediated knockdown of AKT1 also inhibited the degradation of HuR in response to heat shock (Fig. 6C). Exposure to 43°C increased the phosphorylation of AKT1 in a time-dependent manner, without increasing the level of AKT1, indicating the activation of AKT1 by heat shock (Fig. 6D). The Akt inhibitor prevented serine phosphorylation of HuR in response to heat shock (Fig. 6E). HuR also interacted with phosphoAKT1 in response to heat shock, which was abrogated by treatment with Akt inhibitor, suggesting that activated AKT1 interacted with HuR under heat shock (Fig. 6F). WT HuR interacted with phosphoAKT1 while the S100A mutant HuR failed to do so, showing S100 as the target for AKT1-mediated phosphorylation in response to heat shock (Fig. 6G).

As AKT1-mediated phosphorylation of S100 would be dependent on recognition of the substrate motif RXXS/T by AKT1, we disrupted this motif in HuR by mutating R97 to A. The HuR R97A mutant did not get degraded under heat shock, indicating the importance of the RXXS/T motif for substrate recognition by the kinase (Fig. 6H). The R97A mutant also did not undergo serine phosphorylation under heat shock, further demonstrating the requirement of the AKT1 recognition motif for serine phosphorylation of HuR under heat shock (Fig. 6I). Furthermore, the R97A mutant HuR failed to interact with phosphoAKT1 in response to heat shock (Fig. 6J). Together, these findings demonstrated the presence of S100 within a bona fide AKT1 substrate motif in HuR and AKT1 as the kinase phosphorylating S100 under heat shock.

Finally we checked whether AKT1-mediated phosphorylation of HuR was necessary for interaction of HuR with TRIM21 in response to heat shock. Treatment with the Akt inhibitor drastically reduced TRIM21 interaction with HuR in heat-stressed cells (Fig. 6K). siRNA-mediated knockdown of AKT1 also prevented the interaction of HuR with TRIM21 in response to heat shock (Fig. 6L). Finally, the R97A mutant also failed to interact with TRIM21 under heat shock whereas the WT HuR interacted efficiently (Fig. 6M). Together, these observations showed that AKT1-mediated phosphorylation of S100 in HuR allowed the binding of TRIM21 to HuR, leading to polyubiquitination of K182 and subsequent degradation of HuR in response to heat shock.

Post-translational modification (PTM) has emerged as a central regulator of the abundance and functions of RBPs, thereby exerting important effects on the post-transcriptional gene regulatory networks governed by the RBPs³⁵. Importantly, PTMs connect RBPs to multiple signal transduction pathways in cells, especially in response to stress signals, a connection that was hitherto mostly unexplored. This establishes the involvement of RBPs with the cellular signaling pathways, whose outcomes were previously considered to be mainly transcriptional in nature^36,37. Abnormal PTM patterns of RBPs can therefore lead to the alteration of their physiological role and has been associated with the pathophysiology of various disease conditions ^38–41.

PTMs of the RBP HuR have been shown to play especially important roles in regulating the abundance and function of HuR in response to various stimuli²⁴. While HuR is predominantly nuclear in unstimulated cells, it undergoes nuclear-cytoplasmic translocation in response to various stimuli, including stress signals, inflammatory agonists and mitogens^20,42−44. The nuclear-cytoplasmic translocation of HuR is often dependent on PTMs, especially phosphorylation, of specific residues in the HuR nuclear-cytoplasmic shuttling sequence (HNS) located in the hinge region ^43,45−48. Signal-dependent phosphorylation therefore acts as an important mechanism of regulation of HuR subcellular localization and RNA-binding activity.

Although changes in subcellular localization appears to be the major mode of regulation of HuR in response to multiple stimuli, controlled degradation often regulates HuR abundance and function in stress responses. Metabolic stress in cancer cells causes HuR translocation to the cytoplasm where it is targeted by the ubiquitin E3 ligase βTrCP1 for degradation⁴⁹. This is also connected to HuR phosphorylation, as phosphorylation at S318 by PKCα and phosphorylation at S304 by IKKα is required for nuclear-cytoplasmic shuttling and recognition of HuR by βTrCP1 respectively. Nuclear-cytoplasmic shuttling followed by degradation of HuR is also observed in response to genotoxic stress, mediated by the ubiquitin E3 ligase TRIM21^27,50. HuR is also degraded by caspase-mediated cleavage in response to hypoxia and the apoptosis inducer staurosporine^51,52.

Regulated degradation of HuR is also induced in response to heat shock. Degradation of HuR under heat shock was also thought to be connected to phosphorylation of HuR, as the kinase CHK2, which phosphorylates HuR on S88, S100 and T118 upon H₂O₂ treatment, was found to protect HuR against degradation in heat-stressed cells²⁶. HuR degradation under heat shock is also promoted by arginylation of HuR on D15, which was also required for its phosphorylation under heat shock⁵³. Knockdown of the ring-finger domain containing E3 ubiquitin ligase Pirh2, which was found as an interacting partner of HuR, appeared to stabilize HuR protein under heat shock⁵⁴. However, neither the ubiquitination of the K182 residue nor a direct interaction between Pirh2 and HuR under heat shock has been shown. Here we have shown that TRIM21 ubiquitinates K182 under heat shock, leading to its degradation. Moreover, TRIM21 enhances cell survival and reduces apoptosis in response to heat shock, an effect which is mediated by the degradation of HuR. TRIM21 overexpression also enhances cell proliferation and reduces apoptosis at physiological temperature, which conforms to reports of TRIM21’s potential tumorigenic role^55–57. Phosphorylation of S100 was found to be necessary for TRIM21 interaction with HuR and its subsequent degradation. Phosphorylation of S100, together with the presence of the adjacent E101 residue, possibly generates a negative patch on the flexible linker between RRMs 1 and 2 of HuR as a recognition site for TRIM21. Multiple amino acid residues in the linker and in the adjacent parts of RRMs 1 and 2 are sites of phosphorylation regulating different functions of HuR such as localization, RNA-binding and splicing regulation²⁴.

We have shown that the kinase AKT1 phosphorylates S100 and the AKT1-mediated phosphorylation of S100 is necessary for recognition by TRIM21 and degradation of HuR under heat shock. AKT1 or protein kinase B (PKB) acts as one of the most important mediators of cellular signal transduction pathways by phosphorylating a range of intracellular proteins⁵⁸. AKT1 is known to regulate global mRNA translation by activating mammalian target of rapamycin (mTOR), which enhances translation initiation by phosphorylating ribosomal protein S6 and eIF4E-BPs⁵⁹. However, the role of AKT1 in transcript-specific post-transcriptional control of gene expression is not known. By demonstrating AKT1 as causing the phosphorylation and consequent degradation of HuR, we have established a firm link between AKT1 and post-transcriptional regulation of gene expression in cells, especially in response to stress signals. AKT1-mediated phosphorylation of S100 in HuR generates the recognition site for TRIM21 which binds and ubiquitinates K182, leading to the proteasomal degradation of HuR, thereby constituting a heat-shock induced “phosphodegron” in HuR (Fig. 7). A phosphodegron is defined as a phosphorylated residue(s) on a protein that directly interacts with an interaction domain in an E3 ubiquitin ligase, thereby linking the substrate to the conjugation machinery⁶⁰. The regulated degradation of HuR mediated by this phosphodegron controls a crucial adaptive mechanism ensuring cell survival and homeostasis in response to thermal stress.

Plasmid Constructs

Wild type (WT) and deletion and point mutants of HuR coding region were cloned in mammalian expression vector pCDNA3.1 with Myc-His tag. TRIM21 expression construct and haemagluttinin (HA)-tagged ubiquitin expression consruct were gifts from Sunit. K. Singh, BHU, Varanasi, India. and S.N. Bhattacharyya, CSIR-IICB, Kolkata, India respectively.

Cell culture, treatment and transfection

MCF7 human breast carcinoma cells (ATCC HTB-22) grown in DMEM with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) were exposed to heat shock at 43°C for 2 h. Cells were treated with 100 µg/ml cyclohexamide (Amresco, Cleveland, OH, USA) or 10 µM MG132 (Sigma Aldrich, St. Louis, MO, USA) for 1 h, or 10 µM Akt Inhibitor (Sigma Aldrich) for 2 h. Cells were transfected with plasmid vectors, siRNAs (siGENOME SMARTpool TRIM21 (Horizon Discovery, UK), or Invitrogen Silencer Select siRNA for Akt and SMARTpool non-targeting siRNA (Horizon Discovery) or Control siRNA (Sigma MissionsiRNA Universal Negative Control) using Lipofectamine2000 or Turbofect (Thermo Fisher) or peiRfect transfection reagent (BioBharati Life Sciences, India).

Immunoblotting

Lysates of MCF7 cells in S10 lysis buffer were resolved by SDS-PAGE, transferred to PVDF membrane (MilliporeSigma, Burlington, MA, USA), and immunoblotted with antibodies anti-HuR (Santa Cruz Biotechnology, Dallas, TX, USA), TRIM21, Ubiquitin, Myc (Cell Signaling Technology, Danvers, MA, USA), His (BioBharati LifeScience), AKT (ProteinTech), phosphoAkt (R&D Systems), phosphoSerine (Santa Cruz Biotechnology), non-immune IgG (BioBharati), HRP conjugated b-actin (Genscript, Piscataway, NJ, USA) and GAPDH (Santa Cruz Biotechnology) antibodies as primary antibodies. HRP conjugated anti-mouse or anti-rabbit (Cell Signaling Technologies) were used as secondary antibodies. Chemiluminescent signal was detected using FemtolucentPlus HRP substrate (Geno Biosciences, St. Louis, MO, USA).

Coimmunoprecipitation

Cells treated with MG132 and exposed to 43°C for 1h, were lysed with Pierce Direct IP Kit lysis buffer (Thermo Fisher). Lysates were immunoprecipitated with anti-HuR and anti-TRIM21 antibodies using Pierce Direct IP Kit following manufacturer’s protocol, followed by immunoblotting using specific antibodies.

Ni-NTA affinity pulldown assay

MCF7 cells overexpressing His-tagged WT or deletion or point mutant HuR proteins were lysed in NT2 buffer with 10 mM imidazole or NT2 buffer supplemented with 5mM NaF and 5mM Na₃VO₄ for phosphoserine and phoshoAkt pulldown assay. Lysates were incubated with Ni-NTA agarose beads (Qiagen, Germany) overnight at 4°C, washed multiple times with NT2 buffer containing 20 mM Imidazole, following which the proteins were eluted in SDS-PAGE gel-loading dye and resolved by SDS–PAGE followed by immunoblotting using appropriate antibodies.

Ubiquitination Assay

Cells co-transfected with HA-ubiquitin and His-tagged HuR WT treated with MG132 and exposed to 43°C were lysed in ubiquitination lysis buffer with 0.2 mM ATP and subjected to Ni-affinity pull down. The precipitates were washed in NT2 buffer with 20 mM imidazole, following which the precipitates were resolved by SDS-PAGE and immunoblotted with anti-ubiquitin and anti-His antibodies.

RNA-coprecipitation assay:

Cells lysed in NT2 buffer supplemented with 10 mM imidazole were subjected to a Ni-NTA pulldown assay as described earlier. After multiple washes, 20% of the beads were used for western blotting and RNA was isolated from the rest using RNAiso Plus reagent (Takara Bio, Japan). Reverse transcription was done with MMLV RT enzyme (Thermo Fisher) and qPCR was performed using target specific primers.

Quantitative PCR

Total cellular RNA was isolated using RNAiso Plus (Takara Bio). cDNA was synthesized using oligo(dT) primers by MMLV reverse transcriptase (Thermo Fisher). Gene specific primers were used for qPCR using Power SYBR Green reagent (Thermo Fisher), in StepOne plus real-time PCR system (Thermo Fisher). GAPDH mRNA level was used for normalization.

MD Simulation and Analysis:

The GROMOS 54A7⁶¹ force field was used for molecular dynamic simulations. GROMACS⁶² was used as the simulation engine and the water type used was SPC/E (simple point charge extended). The favorable ‘pose’ of the two proteins were computed using molecular docking of the two protein structures (as obtained from PDB) by HADDOCK⁶³. The final MD run was carried out in conditions of NPT for a total time of 400 ns. PDB structures were visualized by Visual Molecular Dynamics (VMD)⁶⁴. Contact between the two proteins was derived by the reduction in the total solvent accessible surface area (SASA)⁶⁵ and residues of contact were determined by checking the pairs of residues (in HuR vs TRIM21) that have their α-carbons within 1 nm of each other.

Cell viability and apoptosis assays

MCF7 cells transfected with plasmid construct or siRNA and subjected to 2 h of heat shock at 43°C post 48 h of transfection, were assayed for cell viability by MTT assay (Sigma Aldrich). Caspase 3/7 activity was measured by CaspaseGlo 3/7 assay (Promega, Madison, WI, USA). Apoptosis of cells was estimated by Annexin V/PI staining using AlexaFluor-488 Annexin V/Dead Apoptosis Kit (Thermo Fisher) followed by epifluorescence microscopy.

Morphology analysis

Cell spread area and perimeterwere determined. Phase contrast images of MCF7 cells transfected with TRIM21 expression vector or TRIM21 siRNA and exposed to 2 h of heat shock were captured using Olympus IX81 inverted microscope using Micromanager software. ImageJ (NIH, USA) software was used for cell spread area analysis. The cell spread areas and perimeters of 400 cells from three different biological repeats were calculated and the data was plotted using GraphPad Prism software.

Cell proliferation and colony formation assay

Cells transfected with plasmid constructs/siRNAs were exposed to 2 h of heat shock 48 hours after transfection and returned to 37°C. Cell proliferation was estimated by MTT assay (Sigma Aldrich) at different time points. For colony-forming assay, transfected MCF7 cells were exposed to heat shock following which they were seeded and allowed to form colonies for 14 days. Colonies were stained with crystal violet and CFUs (colony forming units) were counted.

Live Cell Imaging

Live cell confocal fluorescence time-lapse imaging was performed to measure HuR degradation. Cells were seeded in 35 mm glass bottom dishes (SPL Lifesciences, Korea) and transfected with construct expressing EGFP-tagged HuR WT protein. Post 24 h of transfection, time lapse videos were recorded over a period of 2 h in LSM 710 confocal microscope (Carl Zeiss) in an environmental chamber in 5% CO₂ and 37°C or 43 °C. GFP intensity of whole cell was measured using ZEN 2010 software (Carl Zeiss) and plotted against time.

Statistical analysis

All graphical data represent mean ± standard deviation of at least three independent biological replicates. Single, double and triple *, #, $, @, α, β, γ, δ signs signify p-values ≤ 0.05, ≤ 0.01 and ≤ 0.005 (paired two-tailed Student’s t test or one-tailed Student’s t test as applicable) between controls and samples respectively as indicated in the figures.

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Acknowledgements

We thank S. K. Singh (BHU, Varanasi, India) and S. N. Bhattacharyya (CSIR-IICB, Kolkata, India) for providing reagents. We thank Neelanjana Sengupta (IISER Kolkata, India) and her lab members for help with MD simulation and Swarang Pundalik and Ravi Kumar (IISER Kolkata) for reagents.

Funding

This work was supported by a Wellcome Trust-DBT India Alliance intermediate fellowship (WT500139/Z/09/Z) and Science and Engineering Research Board (SERB) Extramural research grant (EMR/2016/003525) to P.S.R.

Author contributions

A.G, S.N and P.S.R conceived the project, S.N and S.R performed experiments, S.N, S.R. and P.S.R analyzed data, P.S.R and S.N wrote the manuscript, P.S.R obtained funding and supervised the project.

Competing interests

The authors declare no conflict of interests.

There is no duality of interest

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An AKT1- and TRIM21-mediated Phosphodegron Controls Proteasomal Degradation of HuR Enabling Cell Survival under Heat Shock

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