A thrombospondin-dependent pathway for a protective ER stress response.

Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas Thbs4(-/-) mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

Repression of p63 and induction of EMT by mutant Ras in mammary epithelial cells.

The p53-related transcription factor p63 is required for maintenance of epithelial cell differentiation. We found that activated forms of the Harvey Rat Sarcoma Virus GTPase (H-RAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes strongly repress expression of ∆Np63α, the predominant p63 isoform in basal mammary epithelial cells. This regulation occurs at the transcriptional level, and a short region of the ∆Np63 promoter is sufficient for repression induced by H-RasV12. The suppression of ∆Np63α expression by these oncogenes concomitantly leads to an epithelial-to-mesenchymal transition (EMT). In addition, the depletion of ∆Np63α alone is sufficient to induce EMT. Both H-RasV12 expression and ∆Np63α depletion induce individual cell invasion in a 3D collagen gel in vitro system, thereby demonstrating how Ras can drive the mammary epithelial cell state toward greater invasive ability. Together, these results suggest a pathway by which RAS and PIK3CA oncogenes induce EMT through regulation of ∆Np63α.

The myocardin-related transcription factor MKL co-regulates the cellular levels of two profilin isoforms.

Megakaryoblastic leukemia (MKL)/serum-response factor (SRF)-mediated gene transcription is a highly conserved mechanism that connects dynamic reorganization of the actin cytoskeleton to regulation of expression of a wide range of genes, including SRF itself and many important structural and regulatory components of the actin cytoskeleton. In this study, we examined the possible role of MKL/SRF in the context of regulation of profilin (Pfn), a major controller of actin dynamics and actin cytoskeletal remodeling in cells. We demonstrated that despite being located on different genomic loci, two major isoforms of Pfn (Pfn1 and Pfn2) are co-regulated by a common mechanism involving the action of MKL that is independent of its SRF-related activity. We found that MKL co-regulates the expression of Pfn isoforms indirectly by modulating signal transducer and activator of transcription 1 (STAT1) and utilizing its SAP-domain function. Unexpectedly, our studies revealed that cellular externalization, rather than transcription of Pfn1, is affected by the perturbations of MKL. We further demonstrated that MKL can influence cell migration by modulating Pfn1 expression, indicating a functional connection between MKL and Pfn1 in actin-dependent cellular processes. Finally, we provide initial evidence supporting the ability of Pfn to influence MKL and SRF expression. Collectively, these findings suggest that Pfn may play a role in a possible feedback loop of the actin/MKL/SRF signaling circuit.

An IFN-γ-stimulated ATF6-C/EBP-ß-signaling pathway critical for the expression of Death Associated Protein Kinase 1 and induction of autophagy.

The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The death-associated protein kinase 1 (DAPK1), an IFN-γ-induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis, and its expression is frequently down-regulated in a number of human tumors. Although the biochemical action of DAPK1 is well understood, mechanisms that regulate its expression are unclear. Previously, we have shown that transcription factor C/EBP-ß is required for the basal and IFN-γ-induced expression of DAPK1. Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/EBP-ß in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing of ATF6 and ERK1/2-mediated phosphorylation of C/EBP-ß are necessary for these interactions. More importantly, IFN-γ failed to activate autophagic response in cells lacking either ATF6 or C/EBP-ß. Consistent with these observations, the Atf6(-/-) mice were highly susceptible to lethal bacterial infections compared with the wild-type mice. These studies not only unravel an IFN signaling pathway that controls cell growth and antibacterial defense, but also expand the role of ATF6 beyond ER stress.

Serum regulation of Id1 expression by a BMP pathway and BMP responsive element.

Immediate early genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP responsive element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad 4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.

A Non-Canonical Hippo Pathway Represses the Expression of ΔNp63.

The p63 transcription factor, a member of the p53 family, plays an oncogenic role in squamous cell carcinomas, while in breast cancers its expression is often repressed. In the canonical conserved Hippo pathway, known to play a complex role in regulating growth of cancer cells, protein kinases MST1/2 and LATS1/2 act sequentially to phosphorylate and inhibit the YAP/TAZ transcription factors. We found that in MCF10A mammary epithelial cells as well as in squamous and breast cancer cell lines, expression of ΔNp63 RNA and protein is strongly repressed by inhibition of the Hippo pathway protein kinases. While MST1/2 and LATS1 are required for p63 expression, the next step of the pathway, namely phosphorylation and degradation of the YAP/TAZ transcriptional activators is not required for p63 repression. This suggests that regulation of p63 expression occurs by a noncanonical version of the Hippo pathway. We identified similarly regulated genes, suggesting the broader importance of this pathway. Interestingly, lowering p63 expression lead to increased YAP protein levels, indicating crosstalk of the YAP/TAZ-independent and -dependent branches of the Hippo pathway. These results, which reveal the intersection of the Hippo and p63 pathways, may prove useful for the control of their activities in cancer cells.

A non-canonical Hippo pathway represses the expression of ΔNp63.

The p63 transcription factor, a member of the p53 family, plays an oncogenic role in squamous cancers, while in breast cancers its expression is often repressed. In the canonical conserved Hippo pathway, known to play a complex role in regulating growth of cancer cells, the protein kinases MST1/2 and LATS1/2 act sequentially to phosphorylate and inhibit the YAP/TAZ transcription factors. We found that in the MCF10A mammary epithelial cell line as well as in squamous and breast cancer cell lines, expression of ΔNp63 RNA and protein is strongly repressed by inhibition of the Hippo pathway protein kinases in a manner that is independent of p53. While MST1/2 and LATS1 are required for p63 expression, the next step of the pathway, namely phosphorylation and degradation of the YAP/TAZ transcriptional activators is not required for repression of p63. This suggests that regulation of p63 expression occurs by a non-canonical version of the Hippo pathway. We additionally identified additional genes that were similarly regulated suggesting the broader importance of this pathway. Interestingly, we observed that experimentally lowering p63 expression leads to increased YAP protein levels, thereby constituting a feedback loop. These results, which reveal the intersection of the Hippo and p63 pathways, may prove useful for the control of their activities in cancer cells. One Sentence Summary: Regulation of p63 expression occurs by a non-canonical version of the Hippo pathway in mammary epithelial, breast carcinoma and head and neck squamous carcinoma cells.

Activation and repression of cellular immediate early genes by serum response factor cofactors.

The induction of expression of many cellular immediate early genes (IEG) involves the transcription factor serum response factor (SRF). Two families of SRF coactivators have also been implicated in IEG induction, the ternary complex factors (TCFs), ELK1, Sap1, and Net, and the myocardin-related factors, MKL1 and MKL2. We found that serum induction of some SRF target genes is preferentially regulated by MKL1/2, whereas others are redundantly activated by both TCFs and MKL1/2. Yet ELK1 can also repress transcription. Binding of ELK1 and MKL1 to SRF has been found to be mutually exclusive in vitro, suggesting that ELK1 could repress expression of IEGs by blocking MKL1 binding. We characterized the in vivo binding of MKL1 and ELK1 to target genes and found an inverse relationship of serum-induced MKL1 binding and serum-decreased ELK1 binding. However, experiments with short hairpin RNA-mediated MKL1/2 depletion and expression of a nuclear MKL1 (N100) variant in stably transfected cells failed to alter ELK1 binding, suggesting that ELK1 binding to target genes is regulated independently of MKL1/2. Nevertheless, we found that short interfering RNA-mediated depletion of TCFs increased target gene expression in cells containing the N100 MKL1 activator, most notably in cells under continuous growth conditions. These results indicate that the TCFs can function both as activators and repressors of target gene expression depending upon the cellular growth conditions.

ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals.

ATF6 is an endoplasmic reticulum (ER) stress-regulated transmembrane transcription factor that activates the transcription of ER molecular chaperones. Upon ER stress, ATF6 translocates from the ER to the Golgi where it is processed to its active form. We have found that the ER chaperone BiP/GRP78 binds ATF6 and dissociates in response to ER stress. Loss of BiP binding correlates with the translocation of ATF6 to the Golgi, which was slowed in cells overexpressing BiP. Two Golgi localization signals (GLSs) were identified in ATF6. Removal of BiP binding sites from ATF6, while retaining a GLS, resulted in its constitutive translocation to the Golgi. These results suggest that BiP retains ATF6 in the ER by inhibiting its GLSs and that dissociation of BiP during ER stress allows ATF6 to be transported to the Golgi.

Stable binding of ATF6 to BiP in the endoplasmic reticulum stress response.

Endoplasmic reticulum (ER) stress-induced activation of ATF6, an ER membrane-bound transcription factor, requires a dissociation step from its inhibitory regulator, BiP. It has been generally postulated that dissociation of the BiP-ATF6 complex is a result of the competitive binding of misfolded proteins generated during ER stress. Here we present evidence against this model and for an active regulatory mechanism for dissociation of the complex. Contradictory to the competition model that is based on dynamic binding of BiP to ATF6, our data reveal relatively stable binding. First, the complex was easily isolated, in contrast to many chaperone complexes that require chemical cross-linking. Second, ATF6 bound at similar levels to wild-type BiP and a BiP mutant form that binds substrates stably because of a defect in its ATPase activity. Third, ER stress specifically induced the dissociation of BiP from ER stress transducers while the competition model would predict dissociation from any specific substrate. Fourth, the ATF6-BiP complex was resistant to ATP-induced dissociation in vitro when isolated without detergents, suggesting that cofactors stabilize the complex. In favor of an active dissociation model, one specific region within the ATF6 lumenal domain was identified as a specific ER stress-responsive sequence required for ER stress-triggered BiP release. Together, our results do not support a model in which competitive binding of misfolded proteins causes dissociation of the BiP-ATF6 complex in stressed cells. We propose that stable BiP binding is essential for ATF6 regulation and that ER stress dissociates BiP from ATF6 by actively restarting the BiP ATPase cycle.

Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required for serum induction of SRF target genes.

Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that we found strongly activated serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, we assessed its role in serum induction of c-fos and other SRE-regulated genes with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). We found that DN-MKL1 and RNAi specifically blocked SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi required the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduced the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, was nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia had a markedly increased ability to activate SRE reporter genes, suggesting that its activation of SRF target genes may contribute to leukemogenesis.

Pathway Regulation of p63, a Director of Epithelial Cell Fate.

The p53-related gene p63 is required for epithelial cell establishment and its expression is often altered in tumor cells. Great strides have been made in understanding the pathways and mechanisms that regulate p63 levels, such as the Wnt, Hedgehog, Notch, and EGFR pathways. We discuss here the multiple signaling pathways that control p63 expression as well as transcription factors and post-transcriptional mechanisms that regulate p63 levels. While a unified picture has not emerged, it is clear that the fine-tuning of p63 has evolved to carefully control epithelial cell differentiation and fate.

Iron deficiency upregulates Egr1 expression.

Iron-deficient anemia is a prevalent disease among humans. We searched for genes regulated by iron deficiency and its regulated mechanism. cDNA microarrays were performed using Hepa1c1c7 cells treated with 100 µM desferrioxamine (DFO), an iron chelator. Early growth response 1 (Egr1) was upregulated with at least 20-fold increase within 4 h and lasted for 24 h, which was confirmed by qRT-PCR. This activation was not seen by ferric ammonium citrate (FAC). DFO increased the transcriptional activity of Egr1-luc (-604 to +160) and serum response element (SRE)-luc reporters by 2.7-folds. In addition, cycloheximide lowered DFO-induced Egr1 mRNA levels. The upregulation of Egr1 by DFO was accompanied by sustained ERK signals along with phosphorylation of Elk-1. The ERK inhibitor (PD98059) prevented the DFO-induced Egr1 mRNAs. Overexpression of Elk-1 mutant (pElk-1S383A) decreased Egr1 reporter activity. DFO lowered reactive oxygen species (ROS) production and increased caspase 3/7 activity and cell death. DFO-induced iron deficiency upregulates Egr1 in part through transcriptional activation via ERK and Elk-1 signals, which may be important in the regulation of cell death in hepatoma cells. Our study demonstrated that iron depletion controlled the expression of Egr1, which might contribute to decisions about cellular fate in response to iron deficiency.

Filamin A interacts with the coactivator MKL1 to promote the activity of the transcription factor SRF and cell migration.

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor (SRF) that promotes the expression of genes associated with cell proliferation, motility, adhesion, and differentiation-processes that also involve dynamic cytoskeletal changes in the cell. MKL1 is inactive when bound to monomeric globular actin (G-actin), but signals that activate the small guanosine triphosphatase RhoA cause actin polymerization and MKL1 dissociation from G-actin. We found a new mechanism of MKL1 activation that is mediated through its binding to filamin A (FLNA), a protein that binds filamentous actin (F-actin). The interaction of FLNA and MKL1 was required for the expression of MKL1 target genes in primary fibroblasts, melanoma, mammary and hepatocellular carcinoma cells. We identified the regions of interaction between MKL1 and FLNA, and cells expressing an MKL1 mutant that was unable to bind FLNA exhibited impaired cell migration and reduced expression of MKL1-SRF target genes. Induction and repression of MKL1-SRF target genes correlated with increased or decreased MKL1-FLNA interaction, respectively. Lysophosphatidic acid-induced RhoA activation in primary human fibroblasts promoted the association of endogenous MKL1 with FLNA, whereas exposure to an actin polymerization inhibitor dissociated MKL1 from FLNA and decreased MKL1-SRF target gene expression in melanoma cells. Thus, FLNA functions as a positive cellular transducer linking actin polymerization to MKL1-SRF activity, counteracting the known repressive complex of MKL1 and monomeric G-actin.

A novel inhibitory mechanism of MRTF-A/B on the ICAM-1 gene expression in vascular endothelial cells.

The roles of myocardin-related transcription factor A (MRTF-A) and MRTF-B in vascular endothelial cells are not completely understood. Here, we found a novel regulatory mechanism for MRTF-A/B function. MRTF-A/B tend to accumulate in the nucleus in arterial endothelial cells in vivo and human aortic endothelial cells (HAoECs) in vitro. In HAoECs, nuclear localization of MRTF-A/B was not significantly affected by Y27632 or latrunculin B, primarily due to the reduced binding of MRTF-A/B to G-actin and in part, to the low level of MRTF-A phosphorylation by ERK. MRTF-A/B downregulation by serum depletion or transfection of siRNA against MRTF-A and/or MRTF-B induced ICAM-1 expression in HAoECs. It is known that nuclear import of nuclear factor-κB (NF-κB) plays a key role in ICAM-1 gene transcription. However, nuclear accumulation of NF-κB p65 was not observed in MRTF-A/B-depleted HAoECs. Our present findings suggest that MRTF-A/B inhibit ICAM-1 mRNA expression by forming a complex with NF-κB p65 in the nucleus. Conversely, downregulation of MRTF-A/B alleviates this negative regulation without further translocation of NF-κB p65 into the nucleus. These results reveal the novel roles of MRTF-A/B in the homeostasis of vascular endothelium.

Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent.

BACKGROUND: Serum Response Factor (SRF) is a transcription factor that is required for the expression of many genes including immediate early genes, cytoskeletal genes, and muscle-specific genes. SRF is activated in response to extra-cellular signals by its association with a diverse set of co-activators in different cell types. In the case of the ubiquitously expressed immediate early genes, the two sets of SRF binding proteins that regulate its activity are the TCF family of proteins that include Elk1, SAP1 and SAP2 and the myocardin-related MKL family of proteins that include MKL1 and MKL2 (also known as MAL, MRTF-A and -B and BSAC). In response to serum or growth factors these two classes of co-activators are activated by different upstream signal transduction pathways. However, it is not clear how they differentially activate SRF target genes. RESULTS: In order to identify the serum-inducible SRF target genes that are specifically dependent on the MKL pathway, we have performed microarray experiments using a cell line that expresses dominant negative MKL1. This approach was used to identify SRF target genes whose activation is MKL-dependent. Twenty-eight of 150 serum-inducible genes were found to be MKL-dependent. The promoters of the serum-inducible genes were analyzed for SRF binding sites and other common regulatory elements. Putative SRF binding sites were found at a higher rate than in a mouse promoter database but were only identified in 12% of the serum-inducible promoters analyzed. Additional partial matches to the consensus SRF binding site were found at a higher than expected rate in the MKL-dependent gene promoters. The analysis for other common regulatory elements is discussed. CONCLUSIONS: These results suggest that a subset of immediate early and SRF target genes are activated by the Rho-MKL pathway. MKL may also contribute to the induction of other SRF target genes however its role is not essential, possibly due to other activation mechanisms such as MAPK phosphorylation of TCFs.

Fra-1 regulation of Matrix Metallopeptidase-1 (MMP-1) in metastatic variants of MDA-MB-231 breast cancer cells.

Matrix Metallopeptidase 1 (MMP-1) expression has repeatedly been correlated to tumorigenesis and metastasis.  Yet, MMP-1 regulation in a metastatic context remains largely unknown.  Here we confirm differential MMP-1 expression in mammary carcinoma cells with varied metastatic potentials. We show that MMP-1 expression is regulated by an AP-1 element in its promoter in highly metastatic MDA-MB-231 mammary carcinoma cell derivatives.  Fra-1, an AP-1 family transcription factor, differentially binds this element in highly metastatic cells compared to low metastatic cells and is required for MMP-1 expression.  Overexpression of Fra-1 also caused increased MMP-1 expression. Fra-1 mRNA levels are unchanged in the cell variants, however its protein levels are higher in the metastatic cells. While there was no change in Fra-1 protein degradation rates, protein synthesis of Fra-1 was increased in the metastatic cell variant. These results demonstrate that Fra-1 and MMP-1 levels are differentially regulated in metastatic cell variants at the level of Fra-1 protein translation. Consistent with the importance of Fra-1 for tumor growth, we found that Fra-1 overexpression was sufficient to increase cell motility and anchorage independent growth.  These results suggest that increased Fra-1 translation is critical for regulation of MMP-1 and tumor cell metastasis.

Depletion of the transcriptional coactivators megakaryoblastic leukaemia 1 and 2 abolishes hepatocellular carcinoma xenograft growth by inducing oncogene-induced senescence.

Megakaryoblastic leukaemia 1 and 2 (MKL1/2) are coactivators of the transcription factor serum response factor (SRF). Here, we provide evidence that depletion of MKL1 and 2 abolishes hepatocellular carcinoma (HCC) xenograft growth. Loss of the tumour suppressor deleted in liver cancer 1 (DLC1) and the subsequent activation of RhoA were prerequisites for MKL1/2 knockdown-mediated growth arrest. We identified oncogene-induced senescence as the molecular mechanism underlying the anti-proliferative effect of MKL1/2 knockdown. MKL1/2 depletion resulted in Ras activation, elevated p16 expression and hypophosphorylation of the retinoblastoma (Rb) protein in DLC1-deficient HCC cells. Interestingly, reconstitution of HuH7 HCC cells with DLC1 also induced senescence. Evaluation of the therapeutic efficacy of MKL1/2 knockdown in vivo revealed that systemic treatment of nude mice bearing HuH7 tumour xenografts with MKL1/2 siRNAs complexed with polyethylenimine (PEI) completely abolished tumour growth. The regression of the xenografts was associated with senescence. Importantly, PEI-complexed MKL1 siRNA alone was sufficient for complete abrogation of HCC xenograft growth. Thus, MKL1/2 represent promising novel therapeutic targets for the treatment of HCCs characterized by DLC1 loss.

Serum-induced phosphorylation of the serum response factor coactivator MKL1 by the extracellular signal-regulated kinase 1/2 pathway inhibits its nuclear localization.

Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related coactivator of the serum response factor (SRF) transcription factor, which has an integral role in differentiation, migration, and proliferation. Serum induces RhoA-dependent translocation of MKL1 from the cytoplasm to the nucleus and also causes a rapid increase in MKL1 phosphorylation. We have mapped a serum-inducible phosphorylation site and found, surprisingly, that its mutation causes constitutive localization to the nucleus, suggesting that phosphorylation of MKL1 inhibits its serum-induced nuclear localization. The key site, serine 454, resembles a mitogen-activated protein kinase phosphorylation site, and its modification was blocked by the MEK1 inhibitor U0126, implying that extracellular signal-regulated kinase 1/2 (ERK1/2) is the serum-inducible kinase that phosphorylates MKL1. Previous results indicated that G-actin binding to MKL1 promotes its nuclear export, and we found that MKL1 phosphorylation is required for its binding to actin, explaining its effect on localization. We propose a model in which serum induction initially stimulates MKL1 nuclear localization due to a decrease in G-actin levels, but MKL1 is then downregulated by nuclear export due to ERK1/2 phosphorylation.

ER stress signaling by regulated proteolysis of ATF6.

ATF6 is an endoplasmic reticulum (ER) membrane-anchored transcription factor activated by intramembrane proteolysis in the ER stress response. Upon ER stress, ATF6 is transported from the ER to the Golgi to be processed by site-1 and site-2 proteases. The trafficking is controlled by the ER chaperone BiP/GRP78. Here, we describe the experimental methods that we have used to study of ATF6 regulation in tissue culture cells. These methods were used to investigate several key steps of ATF6 activation in the ER stress response including binding and dissociation of BiP to ATF6, translocation from the ER to the Golgi and cleavage in the Golgi. In addition, luciferase reporter assays were a sensitive way to monitor ER stress and ATF6 activation. These methods were not only useful for the study ATF6 and the ER stress response, they might also help to elucidate the roles of the ER stress response in a number of human diseases involving misfolded proteins and in the differentiation of secretory tissues which require higher ER folding capacities.