Subcellular Localization In Response Research Articles

Conditional fluorescent mouse translocation reporters for ERK1/2 and AKT signaling

Understanding how cells activate intracellular signaling pathways in response to external signals, such as growth factors, is a longstanding goal of cell and developmental biology. Recently, live-cell signaling reporters have greatly expanded our understanding of signaling dynamics in response to wide-ranging stimuli and chemical or genetic perturbation, both ex vivo (cell lines) and in vivo (whole embryos or animals). Among the many varieties of reporter systems, translocation reporters that change sub-cellular localization in response to pathway activation have received considerable attention for their ease of use compared to FRET systems and favorable response times compared to transcriptional reporters. We reasoned that mouse reporter lines expressed in a conditional fashion would be a useful addition to the arsenal of mouse genetic tools, as such lines remain undeveloped despite widespread use of these sensors. We therefore created and validated two novel mouse reporter lines at the ROSA26 locus. One expresses an ERK1/2 pathway reporter and a nuclear marker from a single transcript, while the second additionally expresses an AKT reporter in order to simultaneously interrogate both pathways.

AMPK-related protein kinase ARK5 regulates subcellular localization of RNA-binding protein hnRNP A1 during hypertonic stress

The heterogeneous nuclear ribonucleoprotein hnRNP A1 is a nucleocytoplasmic-shuttling RNA-binding protein that plays an important role in nucleic acid metabolism and gene expression regulation. The function of hnRNP A1 is determined in part by its specific location within the cell. Although some work has been done to elucidate the signaling pathways that regulate the cellular localization of hnRNP A1, the precise mechanism(s), including physiological and pathophysiological conditions that alter hnRNP A1 localization, are not known. We previously conducted an unbiased RNAi-based kinome-wide screen to identify kinases that regulate hnRNP A1 localization during hypertonic stress. One of the hits from this screen is AMPK-related protein kinase 5 (ARK5). Here, we validate ARK5 as the kinase responsible for controlling hnRNP A1 subcellular localization in response to hypertonic stress. We find using immunoprecipitation and in vitro kinase assay methods that ARK5 directly interacts with and phosphorylates hnRNP A1 on serine residues within the F-peptide region. We further show that the M9 motif of hnRNP A1 is essential for the ARK5-hnRNP A1 interaction and subsequent phosphorylation. In addition, the silencing of ARK5 increases the expression of antiapoptotic protein Bcl-xL and consequently delays caspase activation during hypertonic stress. Our results indicate that ARK5 phosphorylates hnRNP A1 and regulates its subcellular localization during hypertonic stress.

AU-Rich Element RNA Binding Proteins: At the Crossroads of Post-Transcriptional Regulation and Genome Integrity.

Genome integrity must be tightly preserved to ensure cellular survival and to deter the genesis of disease. Endogenous and exogenous stressors that impose threats to genomic stability through DNA damage are counteracted by a tightly regulated DNA damage response (DDR). RNA binding proteins (RBPs) are emerging as regulators and mediators of diverse biological processes. Specifically, RBPs that bind to adenine uridine (AU)-rich elements (AREs) in the 3′ untranslated region (UTR) of mRNAs (AU-RBPs) have emerged as key players in regulating the DDR and preserving genome integrity. Here we review eight established AU-RBPs (AUF1, HuR, KHSRP, TIA-1, TIAR, ZFP36, ZFP36L1, ZFP36L2) and their ability to maintain genome integrity through various interactions. We have reviewed canonical roles of AU-RBPs in regulating the fate of mRNA transcripts encoding DDR genes at multiple post-transcriptional levels. We have also attempted to shed light on non-canonical roles of AU-RBPs exploring their post-translational modifications (PTMs) and sub-cellular localization in response to genotoxic stresses by various factors involved in DDR and genome maintenance. Dysfunctional AU-RBPs have been increasingly found to be associated with many human cancers. Further understanding of the roles of AU-RBPS in maintaining genomic integrity may uncover novel therapeutic strategies for cancer.

Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A membrane translocation switch

Molecular switches that respond to a biochemical stimulus in cells have proven utility as a foundation for developing molecular sensors and actuators that could be used to address important biological questions. Developing a molecular switch unfortunately remains difficult as it requires elaborate coordination of sensing and actuation mechanisms built into a single molecule. Here, we rationally designed a molecular switch that changes its subcellular localization in response to an intended stimulus such as an activator of protein kinase A (PKA). By arranging the sequence for Kemptide in tandem, we designed a farnesylated peptide whose localization can dramatically change upon phosphorylation by PKA. After testing a different valence number of Kemptide as well as modulating the linker sequence connecting them, we identified an efficient peptide switch that exhibited dynamic translocation between plasma membranes and internal endomembranes in a PKA activity dependent manner. Due to the modular design and small size, our PKA switch can have versatile utility in future studies as a platform for visualizing and perturbing signal transduction pathways, as well as for performing synthetic operations in cells.

Abstract 2650: Characterizing CHCHD2 subcellular localization in response to hypoxia and redox perturbations in glioblastoma cells

Abstract BACKGROUND: Glioblastoma (GBM) is the most common and malignant form of primary brain tumor in adults and remains incurable. Inter-organelle communication between mitochondria and nuclei is a potential compensatory signaling pathway that may contribute to therapeutic resistance and cellular plasticity in the face of a hypoxic tumor microenvironment. Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) is a protein that colocalizes to the mitochondrial inter-membrane space and the nucleus, where it exhibits hypoxia-sensitive transcription factor capabilities. We demonstrated previously that CHCHD2 knockout in hypoxic U87 GBM cells harboring epidermal growth factor receptor (EGFR) variant III (U87vIII) abrogated the increase in proliferation and invasion exhibited by U87vIII WT cells expressing CHCHD2. The C-terminus of CHCHD2 contains redox-sensitive cysteines amenable to thiol/disulfide formation, which determine protein folding. The objective of this study was to characterize the effect of hypoxia and cellular redox status on CHCHD2 subcellular localization. We hypothesized that reducing the C-terminal CX9C motif would induce CHCHD2 mitochondrial export and nuclear accumulation. METHODS: U87 and U87vIII cells were incubated in standard culture oxygen conditions (~21% O2), 7% O2, 4% O2, and pathophysiological hypoxia (1% O2). Confocal immunofluorescence microscopy was used to observe CHCHD2 subcellular localization, and nuclear fluorescence intensity was quantified using Axiovision software. Western blot of whole cell lysates was used to quantify CHCHD2 protein levels in response to decreasing oxygen tensions. Chemical reduction of cells was achieved using dithiothreitol (DTT) at designated concentrations and time courses. RESULTS: The amount of nuclear CHCHD2 did not differ between 21% and 7% O2. Incubation in 1% O2 increased the amount of nuclear CHCHD2 in both U87 and U87vIII cells, with the latter consistently exhibiting greater amounts of nuclear CHCHD2 at all oxygen tensions. Total CHCHD2 protein levels in U87 cells were maximal at 4% O2, but reduced at 1% O2, a trend also observed in U87vIII cells. Chemical reduction with 1 mM DTT for either 15 min or 24 h induced significantly greater CHCHD2 nuclear accumulation in U87vIII cells compared to U87. Increasing DTT dose 10-fold resulted in rapid accumulation of CHCHD2 in nuclei after 15 min, and complete disappearance from mitochondria after 30 min. CONCLUSIONS: Hypoxia and chemical reduction are capable of driving CHCHD2 translocation from mitochondria to the nucleus in U87 GBM cells, which is enhanced in the presence of the EGFRvIII mutant. Ongoing work to expand our understanding of how CHCHD2 protein folding governs protein localization and function may inspire novel therapeutic strategies to undermine mitonuclear signaling in GBM. Citation Format: Jan C. Lumibao, H. Rex Gaskins. Characterizing CHCHD2 subcellular localization in response to hypoxia and redox perturbations in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2650.

Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1-related protein kinase 2.10 in response to osmotic stress.

SNF1-related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA-dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA-non-activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S-segment is involved in the regulation of dehydrin subcellular localization in response to stress.

Substrate Stiffness Influences the Time Dependence of CTGF Protein Expression in Müller Cells.

Following ocular trauma and retinal detachment, gliotic changes in the retina may develop over the subsequent month, a process known as PVR (proliferative vitreoretinopathy). There have been no successful therapeutic interventions to inhibit PVR. The protein CTGF (Connective Tissue Growth Factor) has been associated with retinal PVR and other fibrotic diseases of the retina in clinical studies but the mechanistic link between different pathologies and retinal gliosis has not been determined. In addition, CTGF has been previously noted to be associated, in some cases, with YAP/TAZ (Yes-associated protein and Tafazzin protein complex), transcriptional regulatory proteins that change subcellular localization in response to mechanical cues, such as the stiffness of the underlying material. We have previously shown that the mRNA for CTGF is markedly (100-fold) upregulated in retinal Müller cells grown on soft substrates. In order to evaluate if the mechanism by which mechanotransduction modulating CTGF production in retinal Müller cells involves the YAP/TAZ complex, this study tests the influence of substrate stiffness on the time dependence of CTGF protein expression, as well as subcellular localization of YAP/TAZ using a conditionally-immortalized mouse retinal Müller cell line plated on laminin-coated, polyacrylamide substrates of varying elastic modulus. Changes were assayed using immunohistochemistry and ELISA (Enzyme-Linked ImmunoSorbent Assay). In retinal Müller cells, the relationship between elastic modulus and the pattern of CTGF protein expression was bimodal, with CTGF levels rising more rapidly for cells on hard substrates and more slowly for cells grown on soft substrates. In addition, nuclear localization of YAP/TAZ corresponded directly to the maximum CTGF expression.

A sucrose transporter-interacting protein disulphide isomerase affects redox homeostasis and links sucrose partitioning with abiotic stress tolerance.

Sucrose accumulation in leaves in response to various abiotic stresses suggests a specific role of this disaccharide for stress tolerance and adaptation. The high-affinity transporter StSUT1 undergoes substrate-induced endocytosis presenting the question as to whether altered sucrose accumulation in leaves in response to stresses is also related to enhanced endocytosis or altered activity of the sucrose transporter. StSUT1 is known to interact with several stress-inducible proteins; here we investigated whether one of the interacting candidates, StPDI1, affects its subcellular localization in response to stress: StPDI1 expression is induced by ER-stress and salt. Both proteins, StSUT1 and StPDI1, were found in the detergent resistant membrane (DRM) fraction, and this might affect internalization. Knockdown of StPDI1 expression severely affects abiotic stress tolerance of transgenic potato plants. Analysis of these plants does not reveal modified subcellular localization or endocytosis of StSUT1, but rather a disturbed redox homeostasis, reduced detoxification of reactive oxygen species and effects on primary metabolism. Parallel observations with other StSUT1-interacting proteins are discussed. The redox status in leaves seems to be linked to the sugar status in response to various stress stimuli and to play a role in stress tolerance.

Bik subcellular localization in response to oxidative stress induced by chemotherapy, in Two different breast cancer cell lines and a Non-tumorigenic epithelial cell line.

Cancer chemotherapy remains one of the preferred therapeutic modalities against malignancies despite its damaging side effects. An expected outcome while utilizing chemotherapy is apoptosis induction. This is mainly regulated by a group of proteins known as the Bcl-2 family, usually found within the endoplasmic reticulum or the mitochondria. Recently, these proteins have been located in other sites and non-canonic functions have been unraveled. Bik is a pro-apoptotic protein, which becomes deregulated in cancer, and as apoptosis is associated with oxidative stress generation, our objective was to determine the subcellular localization of Bik either after a direct oxidative insult due to H2 O2 , or indirectly by cisplatin, an antineoplastic agent. Experiments were performed in two human transformed mammary gland cell lines MDA-MB-231 and MCF-7, and one non-tumorigenic epithelial cell line MCF-10A. Our results showed that in MCF-7, Bik is localized within the cytosol and that after oxidative stress treatment it translocates into the nucleus. However, in MDA-MB-231, Bik localizes in the nucleus and translocates to the cytosol. In MCF10A Bik did not change its cellular site after either treatment. Interestingly, MCF10A were more resistant to cisplatin than transformed cell lines. This is the first report showing that Bik is located in different cellular compartments depending on the cancer stage, and it has the ability to change its subcellular localization in response to oxidative stress. This is associated with increased sensitivity when exposed to toxic agents, thus rendering novel opportunities to study new therapeutic targets allowing the development of more active and less harmful agents.

Genome-wide single-cell-level screen for protein abundance and localization changes in response to DNA damage in S. cerevisiae

An effective response to DNA damaging agents involves modulating numerous facets of cellular homeostasis in addition to DNA repair and cell-cycle checkpoint pathways. Fluorescence microscopy-based imaging offers the opportunity to simultaneously interrogate changes in both protein level and subcellular localization in response to DNA damaging agents at the single-cell level. We report here results from screening the yeast Green Fluorescent Protein (GFP)-fusion library to investigate global cellular protein reorganization on exposure to the alkylating agent methyl methanesulfonate (MMS). Broad groups of induced, repressed, nucleus- and cytoplasm-enriched proteins were identified. Gene Ontology and interactome analyses revealed the underlying cellular processes. Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins. An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed. Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF. We conclude that cellular responses can navigate different routes according to the extent of damage, relying on both expression and localization changes of specific proteins.

CDK1 interacts with RARγ and plays an important role in treatment response of acute myeloid leukemia

Alterations in cell cycle pathways and retinoic acid signaling are implicated in leukemogenesis. However, little is known about the roles of cyclin-dependent kinases (CDKs) in treatment response of leukemia. In this study, we observed that CDK1 expression was significantly higher in bone marrow from 42 patients with acute myeloid leukemia (AML) at recurrence than that at first diagnosis (p = 0.04). AML patients had higher level of nuclear CDK1 in their leukemic blasts tended to have poorer clinical outcome compared with those with lower levels. We showed that CDK1 function is required for all-trans retinoic acid (ATRA) to achieve the optimal effect in U-937 human leukemic cells. CDK1 modulates the levels of P27kip and AKT phosphorylation in response to ATRA treatment. Further, we show, for the first time, that RARγ in concert with ATRA regulates protein levels of CDK1 and its subcellular localization. The regulation of the subcellular content of CDK1 and RARγ by ATRA is an important process for achieving an effective response in treatment of leukemia. RARγ and CDK1 form a reciprocal regulatory circuit in the nucleus and influence the function and protein stability of each other and the level of P27kip protein. In addition, expression of wee1 kinase and Cdc25A/C phosphatases also coincide with CDK1 expression and its subcellular localization in response to ATRA treatment. Our study reveals a novel mechanism by which CDK1 and RARγ coordinate with ATRA to influence cell cycle progression and cellular differentiation.

Phosphorylation by Casein Kinase 1 Regulates Tonicity-induced Osmotic Response Element-binding Protein/Tonicity Enhancer-binding Protein Nucleocytoplasmic Trafficking

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, is the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Although it is well documented that the subcellular localization and transactivation activity of OREBP/TonEBP are tightly regulated by extracellular tonicity, the molecular mechanisms involved remain elusive. Here we show that nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by the dual phosphorylation of Ser-155 and Ser-158. Alanine scanning mutagenesis revealed that Ser-155 is an essential residue that regulates OREBP/TonEBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser-155 and Ser-158 of OREBP/TonEBP are both phosphorylated in living cells under hypotonic conditions. In vitro phosphorylation assays further suggest that phosphorylation of the two serine residues proceeds in a hierarchical manner with phosphorylation of Ser-155 priming the phosphorylation of Ser-158 and that these phosphorylations are essential for nucleocytoplasmic trafficking of the transcription factor. Finally, we have shown that the pharmacological inhibition of casein kinase 1 (CK1) abolishes the phosphorylation of Ser-158 and impedes OREBP/TonEBP nuclear export and that recombinant CK1 phosphorylates Ser-158. Knockdown of CK1alpha1L, a novel isoform of CK1, inhibits hypotonicity-induced OREBP/TonEBP nuclear export. Together these data highlight the importance of Ser-155 and Ser-158 in the nucleocytoplasmic trafficking of OREBP/TonEBP and indicate that CK1 plays a major role in regulating this process.

Visual Stimulation Activates ERK in Synaptic and Somatic Compartments of Rat Cortical Neurons with Parallel Kinetics

BackgroundExtracellular signal-regulated kinase (ERK) signalling pathway plays a crucial role in regulating diverse neuronal processes, such as cell proliferation and differentiation, and long-term synaptic plasticity. However, a detailed understanding of the action of ERK in neurons is made difficult by the lack of knowledge about its subcellular localization in response to physiological stimuli. To address this issue, we have studied the effect of visual stimulation in vivo of dark-reared rats on the spatial-temporal dynamics of ERK activation in pyramidal neurons of the visual cortex.Methodology/Principal FindingsUsing immunogold electron microscopy, we show that phosphorylated ERK (pERK) is present in dendritic spines, both at synaptic and non-synaptic plasma membrane domains. Moreover, pERK is also detected in presynaptic axonal boutons forming connections with dendritic spines. Visual stimulation after dark rearing during the critical period causes a rapid increase in the number of pERK-labelled synapses in cortical layers I–II/III. This visually-induced activation of ERK at synaptic sites occurs in pre- and post-synaptic compartments and its temporal profile is identical to that of ERK activation in neuronal cell bodies.Conclusions/SignificanceVisual stimulation in vivo increases pERK expression at pre- and post-synaptic sites of axo-spinous junctions, suggesting that ERK plays an important role in the local modulation of synaptic function. The data presented here support a model in which pERK can have early and late actions both centrally in the cell nucleus and peripherally at synaptic contacts.

Mouse Rab23 regulates Hedgehog signaling from Smoothened to Gli proteins

Sonic hedgehog (Shh) signaling is required for the growth and patterning of many tissues in vertebrate embryos, but important aspects of the Shh signal transduction pathway are poorly understood. For example, the vesicle transport protein Rab23 is a cell autonomous negative regulator of Shh signaling, but the process affected by Rab23 has not been defined. Here, we demonstrate that Rab23 acts upstream of Gli transcription factors in patterning neural cell types in the spinal cord. Double mutant analysis indicates that the primary target of Rab23 is the Gli2 activator and that Rab23 and Gli3 repressor have additive effects on patterning. Analysis of Gli3 protein suggests that Rab23 also has a role in promoting the production of Gli3 repressor. Although the membrane proteins Patched and Smoothened change subcellular localization in response to Shh, double mutant analysis demonstrates that Rab23 does not work through either Patched or Smoothened. Instead, Rab23 appears to regulate subcellular localization of essential components of the Hedgehog pathway that act downstream of Smoothened and upstream of Gli proteins.

Mechanism of Diacylglycerol-induced Membrane Targeting and Activation of Protein Kinase Cδ

The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2-diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins. Although recent reports have indicated that C1A and C1B domains of conventional PKCs play different roles in their DAG-mediated membrane binding and activation, the individual roles of C1A and C1B domains in the DAG-mediated activation of novel PKCs have not been fully understood. In this study, we determined the roles of C1A and C1B domains of PKCdelta by means of in vitro lipid binding analyses and cellular protein translocation measurements. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCdelta have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCdelta mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKCdelta. The studies also indicated that an anionic residue, Glu(177), in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidylserine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKCdelta and mutants showed that because of its phosphatidylserine specificity PKCdelta preferentially translocated to the plasma membrane under the conditions in which DAG is randomly distributed among intracellular membranes of HEK293 cells. Collectively, these results provide new insight into the differential roles of C1 domains in the DAG-induced membrane activation of PKCdelta and the origin of its specific subcellular localization in response to DAG.

Molecular Cloning and Characterization of the Human Diacylglycerol Kinase β (DGKβ) Gene: ALTERNATIVE SPLICING GENERATES DGKβ ISOTYPES WITH DIFFERENT PROPERTIES

Diacylglycerol kinases are key modulators of levels of diacylglycerol, a second messenger involved in a variety of cellular responses to extracellular stimuli. A number of diacylglycerol kinases encoded by separate genes are present in mammalian genomes. We have cloned cDNAs encoding several isoforms of the human homologue of the rat diacylglycerol kinase beta gene and characterized two such isoforms that differ at their carboxyl terminus through alternative splicing and the usage of different polyadenylation signals. Quantitative analysis of gene expression in a panel of human tissue cDNAs revealed that transcripts corresponding to both isoforms are co-expressed in central nervous system tissues and in the uterus, with one variant being expressed at relatively higher levels. As green fluorescent protein fusions, the two isoforms displayed localization to different subcellular compartments, with one variant being associated with the plasma membrane, while the other isoform was predominantly localized within the cytoplasm. Differences were also observed in their subcellular localization in response to phorbol ester stimulation. Enzymatic assays demonstrated that the two isoforms display comparable diacylglycerol kinase activities. Therefore, the human diacylglycerol kinase beta gene can generate several enzyme isoforms, which can display different expression levels and subcellular localization but similar enzymatic activities in vitro.