One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.

Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval.

The cation-independent mannose 6-phosphate receptor (CIMPR) cycles between the trans-Golgi network (TGN) and endosomes to mediate sorting of lysosomal hydrolases. The endosome-to-TGN retrieval of the CIMPR requires the retromer complex. Genetic, biochemical and structural data support the hypothesis that the retromer can directly bind to the tail of the CIMPR, to sort the CIMPR into vesicles and tubules for retrieval to the TGN. Presently, however, no known retromer sorting motif in the tail of the CIMPR has been identified. Using CD8-reporter proteins carrying the cytoplasmic tail of the CIMPR we have systematically dissected the CIMPR tail to identify a novel, conserved aromatic-containing sorting motif that is critical for the endosome-to-TGN retrieval of the CIMPR and for the interaction with retromer and the clathrin adaptor AP-1.

Role of TCR-induced extracellular signal-regulated kinase activation in the regulation of early IL-4 expression in naive CD4+ T cells.

Although extracellular signal-regulated kinase (Erk) activation influences IL-4 production in various experimental systems, its role during Th differentiation is unclear. In this study, we show that Erk plays a critical role in IL-4 expression during TCR-induced Th differentiation of naive CD4(+) T cells. Stimulation of CD4(+) T cells with a high affinity peptide resulted in sustained Erk activation and Th1 differentiation. However, reduction of Erk activity led to a dramatic increase in IL-4 production and Th2 generation. Analysis of RNA and nuclear proteins of CD4(+) T cells 48 h after stimulation revealed that this was due to early IL-4 expression. Interestingly, transient Erk activation resulted in altered AP-1 DNA binding activity and the induction of an AP-1 complex that was devoid of Fos protein and consisted of Jun-Jun dimers. These data show that in the presence of a strong TCR signal, IL-4 expression can be induced in naive CD4(+) T cells by altering the strength of Erk activation. In addition, these data suggest that TCR-induced Erk activation is involved in the regulation of IL-4 expression by altering the composition of the AP-1 complex and its subsequent DNA binding activity.

Expression and purification of recombinant human c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro.

c-Fos and c-Jun are members of the AP-1 family of transcriptional activators that regulate the expression of genes during cell proliferation. To facilitate in vitro studies of mechanisms of transcriptional activation by c-Jun and c-Fos we developed a method for obtaining recombinant c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro. Full-length human c-Fos and c-Jun were expressed in Escherichia coli. The expression of c-Fos was dependent on a helper plasmid that encodes rare (Arg)tRNAs. Both over-expressed c-Fos and c-Jun were recovered from inclusion bodies. A c-Fos/c-Jun complex was generated by co-renaturation and purified via a His-tag on the full-length human c-Fos. The resulting c-Fos/c-Jun bound DNA with high affinity and specificity, and activated transcription in a reconstituted human RNA polymerase II transcription system. The availability of active recombinant human c-Fos/c-Jun will allow future biochemical studies of these important transcriptional activators.

Requirement of an AP-1 site in the calcium response region of the involucrin promoter.

Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of the skin. The gene expression of this differentiation marker is induced by elevated extracellular calcium in cultured human keratinocytes. A 3.7-kilobase fragment of this gene contains the necessary elements to drive a luciferase reporter in a calcium-dependent manner. We have sequenced the upstream region of the involucrin promoter and localized a calcium response element that contains an activating protein-1 (AP-1) site (TGAGTCA). Mutation of this site abolished the promoter activation by calcium. Compared with cells grown in 0.03 mm calcium, the binding activity of factors within nuclear extracts from keratinocytes for this AP-1 site was enhanced 3-fold in cells grown in 1.2 mm calcium. Immunoelectrophoretic mobility shift (supershift) assays identified JunD, Fra1, and Fra2 as the major factors that bind to the AP-1 element. Western analysis of the proteins in the nuclear extracts showed that the levels of c-Jun, JunB, JunD, FosB, and Fra2 increased and the levels of c-Fos and Fra1 decreased slightly with calcium treatment. The effect of calcium on the involucrin promoter was enhanced synergistically by phorbol 12-myristate 13-acetate (PMA) in a protein kinase-dependent manner. In conclusion, calcium-regulated involucrin gene expression is mediated at least in part by AP-1 transcription factors.

An efficient method for simultaneous isolation of biologically active transcription factors and DNA.

Transcription factors play a crucial role in gene regulation during different stages of eukaryotic development as well as in controlling various cellular disorders involving the immune system. In order to study the role of cellular DNAs and the effects of certain biologically active regulatory proteins, which can affect gene expression, we have developed a rapid and efficient method for preparing highly purified DNAs as well as nuclear and cytoplasmic proteins, simultaneously. These DNAs and proteins can be effectively analyzed to determine their genetic integrity and binding motifs to specific DNA sequences, respectively. This protocol avoids the drastic use of mechanical shearing of cells, aggressive use of detergents or high speed ultracentrifugation steps, as well as facilitating the ease of collecting samples in a sequential and effective manner with minimal time lapse during processing. Such an approach permits the analysis of a large number of samples in a short time. The current technique uses a non-ionic detergent to isolate nuclei, and obtain the cytosolic extract, a low-ionic strength buffer to wash off the detergent and a high-salt buffer to extract nuclear proteins including transcription factors. The remainder of the cellular products are processed for DNA extraction. This method will be particularly useful to evaluate the time course effects of various cell signal transducing biological modifiers such as cytokines or mitogens, as well as drugs used in therapy, especially in infectious diseases and also in immunological or neoplastic disorders, with minimal physical contact to the laboratory personnel. This rapid DNA and protein isolation method can be widely used in various systems to analyze the modulation of DNA characteristics and transcriptionally active proteins as biomarkers in different human diseases.

Lack of c-Jun activity increases survival to cisplatin.

Antineoplasic agents such as cisplatin and adriamycin execute their pharmacological role by triggering apoptosis. We have studied the mechanism of apoptosis induction by cisplatin and adriamycin. Both drugs activated JNK with slow and persistent kinetics. Adriamycin activated caspase-3 before the rise in JNK activity, while the response to cisplatin occurs hours after JNK activation. The increase in JNK activity was necessary for cisplatin-mediated apoptosis but it was dispensable for adriamycin-induced cell death. Cells derived from c-jun knock out mice were more resistant to cisplatin cell death than normal cells, but no difference was observed in response to adriamycin. Activation of JNK and cell death by cisplatin is mediated by the MEKK1/SEK1 cascade, since expression of dominant negative expression vectors of these kinases blocked both processes. p38 was also activated by cisplatin with similar kinetics as JNK. AP-1 complexes were activated by cisplatin including mainly c-jun/ATF-2 heterodimers suggesting that AP-1-dependent transcription partially mediated cisplatin-induced apoptosis.

Apoptosis during castration-induced regression of the prostate is Fos dependent.

Apoptotic cell death was shown to be accompanied or preceded by an elevated expression of the c-fos protooncogene and DNA binding activity of transcription factor AP-1. We used Fos-deficient mice to study the role of c-Fos during programmed cell death in the prostate. In normal mice apoptosis is induced in the prostate within 2-4 days after castration. Histological features of reduced secretory activity and morphological signs of programmed cell death become obvious. No apparent decrease in secretory activity and no epithelial cell death were observed in Fos-deficient animals after castration. Fragmentation of nuclear DNA was measured by in situ terminal transferase reaction. DNA fragmentation was observed in the prostate epithelium of control mice after castration whereas no similar fragmentation was found in Fos-deficient animals. After castration an AP-1 complex accumulated in the prostate of Fos deficient mice which mainly consists of FosB, Fra-2 and JunD whereas in control animals the AP-1 complex in addition contained c-Fos. Our data strongly suggest that c-Fos is required for programmed cell death of prostate epithelial cells.

Detection and characterization of Sp1 binding activity in human chondrocytes and its alterations during chondrocyte dedifferentiation.

We have detected DNA binding activity for a synthetic oligonucleotide containing an Sp1 consensus sequence in nuclear extracts from human chondrocytes. Changes in the levels of Sp1 oligonucleotide binding activity were examined in nuclear extracts from freshly isolated human chondrocytes, from chondrocytes that had been cultured under conditions that allowed the maintenance of a chondrocyte-specific phenotype on plastic dishes coated with the hydrogel poly(2-hydroxyethyl methacrylate), and from chondrocytes induced to dedifferentiate into fibroblast-like cells by passage in monolayer culture on plastic substrata. It was observed that Sp1 binding was 2-3-fold greater in nuclear extracts from dedifferentiated chondrocytes than in nuclear extracts from either freshly isolated chondrocytes or from cells cultured in suspension. The Sp1 binding activity was specific, since it was competed by unlabeled Sp1 but not by AP1 or AP2. The addition of a polyclonal antibody against Sp1 to nuclear extracts from freshly isolated chondrocytes or to extracts isolated from chondrocytes cultured in monolayer decreased the binding of Sp1 by approximately 85%. However, when the same experiment was carried out with nuclear extracts prepared from cells cultured on poly(2-hydroxyethyl methacrylate)-coated plates, only a very slight inhibition of Sp1 binding was observed. When fragments of the COL2A1 promoter containing putative Sp1 binding sites amplified by polymerase chain reaction were examined, it was found that the amounts of DNA-protein complex formed with nuclear extracts from dedifferentiated chondrocytes were 2-3-fold greater than the amounts formed with nuclear extracts from freshly isolated chondrocytes or from cells cultured in suspension. Quantitation of DNA binding activity by titration experiments demonstrated that nuclear extracts from fibroblast-like cells contained approximately 2-fold greater Sp-1 specific binding activity than nuclear extracts from chondrocytes. The direct role of Sp1 in type II collagen gene transcription was demonstrated by co-transfection experiments of COL2A1 promoter-CAT constructs in Drosophila Schneider line L2 cells that lack Sp1 homologs. This is the first demonstration of Sp1 binding activity in human chondrocytes and of differences in Sp1 DNA binding activity between differentiated and dedifferentiated chondrocytes.

Modulation of JunD.AP-1 DNA binding activity by AP-1-associated factor 1 (AF-1).

AP-1-associated factor 1 (AF-1), is a novel protein complex that dramatically enhances the assembly of JunD-containing dimers onto AP-1 consensus sites. We describe the partial purification of AF-1 from nuclear extracts of the T-cell line MLA 144 by ionic, hydrophobic and gel filtration chromatography. AF-1 is a DNA-binding protein composed of low molecular mass polypeptides of 7-17 kDa that exists in solution as a 34-kDa complex. JunD interactions with DNA are accelerated in the presence of AF-1 through the formation of a true tri-molecular complex with JunD dimers and DNA that assembles much more rapidly on DNA than JunD alone. DNA binding analysis of AF-1 interaction with JunD.AP-1 and DNA shows that AF-1 increases the DNA binding affinity of JunD for AP-1 sites over 100-fold. DNA cleavage footprint analysis of isolated AF-1.JunD DNA complexes shows that the ternary complex makes nearly twice as many contacts with DNA than JunD dimers alone. AF-1 interacts readily, but differentially with Jun homodimers and Jun.Fos heterodimers. These findings distinguish AF-1 as a significant protein-specific modulator of AP-1.JunD in T-cells.

Interactions of the transcription factor AP-1 with the long terminal repeat of different human immunodeficiency virus type 1 strains in Jurkat, glial, and neuronal cells.

Human immunodeficiency virus type 1 (HIV-1) infection of the neuronal and astroglial cells of the central nervous system has been proposed to contribute to HIV-1-associated dementia. Recently it was shown that differences in the nucleotide sequence of the long terminal repeat (LTR) of different HIV-1 strains govern the tissue-specific pattern of viral expression. The LTR from central nervous system-derived HIV-1 strains JR-FL and JR-CSF directs expression in the neurons of transgenic mice, in contrast with the lymphotropic LAI strain. By in vitro footprinting, gel retardation, and methylation interference experiments, we have studied the interactions of host cell proteins from human neuronal, glial, HeLa, and Jurkat T cells with the LTRs from the neurotropic JR-FL and JR-CSF strains, compared with the LAI strain. Proteins belonging to the nuclear receptor family bind with different affinities to variant -352 to -324 sites. Gel supershift assays with Jun and Fos antibodies showed that the AP-1 transcription factor present in the various cell types was unable to recognize the -352 to -324 and -306 to -285 AP-1 putative binding sites. Interestingly, Jun and Fos components of AP-1 interact with the variant TGGCTCA sequence located in the -247 to -222 region of both neurotropic strains. These interactions were cell type specific, since they were detected only with extracts from glial and HeLa cells and not from neuronal or Jurkat cells. Cotransfection experiments further revealed that the -247 to -222 sequence is able to mediate AP-1-induced transcriptional activation in glial and not neuronal cells.

Sphingosylphosphocholine, a signaling molecule which accumulates in Niemann-Pick disease type A, stimulates DNA-binding activity of the transcription activator protein AP-1.

Sphingosylphosphocholine (SPC) is the deacylated derivative of sphingomyelin known to accumulate in neuropathic Niemann-Pick disease type A. SPC is a potent mitogen that increases intracellular free Ca2+ and free arachidonate through pathways that are only partly protein kinase C-dependent. Here we show that SPC increased specific DNA-binding activity of transcription activator AP-1 in electrophoretic mobility-shift assays. Increased DNA-binding activity of AP-1 was detected after only 1-3 min, was maximal after 6 hr, and remained elevated at 12-24 hr. c-Fos was found to be a component of the AP-1 complex. Northern hybridization revealed an increase in c-fos transcripts after 30 min. Since the increase in AP-1 binding activity preceded the increase in c-fos mRNA, posttranslational modifications may be important in mediating the early SPC-induced increases in AP-1 DNA-binding activity. Western analysis detected increases in nuclear c-Jun and c-Fos proteins following SPC treatment. SPC also transactivated a reporter gene construct through the AP-1 recognition site, indicating that SPC can regulate the expression of target genes. Thus, SPC-induced cell proliferation may result from activation of AP-1, linking signal transduction by SPC to gene expression. Since the expression of many proteins with diverse functions is known to be regulated by AP-1, SPC-induced activation of AP-1 may contribute to the pathophysiology of Niemann-Pick disease.

The rat alpha 1B adrenergic receptor gene middle promoter contains multiple binding sites for sequence-specific proteins including a novel ubiquitous transcription factor.

Transcription of the rat alpha 1B adrenergic receptor (alpha 1BAR) gene in the liver is controlled by three promoters that generate three mRNAs. The middle promoter (P2), located between -432 and -813 base pairs upstream from the translation start codon and lacking a TATA box, is responsible for generating the major, 2.7-kilobase mRNA-species expressed in many tissues (Gao, B., and Kunos, G. (1994) J. Biol. Chem. 269, 15762-15767). DNase I footprinting using rat liver nuclear extracts identified three protected regions in P2: footprint I (-432 to -452), footprint II (-490 to -540), and footprint III (-609 to -690). Putative response elements in footprints I and III were not analyzed except the AP2 binding site in footprint III, which could be protected by purified AP2 protein. Footprint II contains four sites corresponding to half of the NF-I consensus sequence, but DNA mobility shift assays indicate that this footprint binds two proteins distinct from NF-I: a ubiquitous CP1-related factor and another novel factor, termed alpha-Adrenergic Receptor Transcription Factor (alpha ARTF), which binds to two separate sites in this region. The alpha ARTF is widely distributed, with the highest amounts found in brain, followed by liver, kidney, lung, and spleen, but no detectable activity in heart. Deletions of alpha ARTF binding sites nearly abolished P2 promoter activity, which suggests that the alpha ARTF is essential for the transcription of the alpha 1BAR gene in most tissues.

Purification and characterization of a multicomponent AP-1.junD complex from T cells. Dependence on a separate cellular factor for enhanced DNA binding activity.

TAP-1 (T-cell AP-1) is a previously identified DNA-binding activity that is rapidly induced in activated T cells in the absence of protein synthesis. This activity has been purified over 2,000-fold from the T-cell line MLA144. Purified TAP-1 is a multicomponent complex composed of 38-kDa and 43-kDa junD polypeptides in association with a separate factor(s), distinct from fos, that partly dissociate from the complex during affinity purification but is required for full TAP-1 DNA-binding activity. When reconstituted with TAP-1, this partly dissociated component strongly enhances the DNA-binding activity of the TAP-1 complex. UV-cross-linking analysis identifies the dissociable component of the TAP-1 complex as a separate class of low molecular mass (23-29-kDa) DNA-binding polypeptide(s). 23-29-kDa polypeptides have been partially purified from nuclear extracts derived from MLA144 that enhance TAP-1 DNA-binding activity over 100-fold and increase its contacts with flanking DNA sequence. These results define TAP-1 as a distinct AP-1.junD-containing complex in T cells whose DNA-binding activity is regulated by the interaction of distinct and separate cellular factor(s).