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A murine cardiac Agtll expression library was screened with an amphipathic helix antibody, and a recombinant representing the C-terminal 194 residues of murine HSP90 (HSP84) was cloned. Both recombinant and native HSP9Os were then found to rapidly convert a basic helix-loop-helix protein (MyoDi) from an inactive to an active conformation, as assayed by sequence-specific DNA binding. The conversion process involves a transient interaction between HSP9O and MyoDl and does not result in the formation of a stable tertiary complex. Conversion does not require ATP and occurs stoichiometrically in a dose-dependent fashion.HSP90 is an abundant, ubiquitous, and highly conserved protein present in most eukaryotic cells. These results provide direct evidence that HSP90 can affect the conformational structure of a DNA-binding protein.Heat shock proteins (HSPs) are thought to aid cells coping with stress, and their expression is induced by heat, heavy metals, pH, and alcohol (reviewed in references 13 and 16). Recent studies have shown that some classes of HSP (HSP60 and HSP70) also function in membrane translocation and folding of proteins in cells that are not under stress (reviewed in reference 13). Synthesis of HSP90 increases in some cells three to fivefold under stress conditions, but it is also abundant in uninduced cells (22). Most of the information acquired so far about the function of HSP90 has been through association, and an activity for the protein has not been described in vitro. HSP90 interacts with several viral oncogene products that display tyrosine kinase activity (4,25,31,39), associates with tubulin and actin (28, 36), stimulates the activity of eukarytotic initiation factor 2 a subunit-specific protein kinase (33), and forms stable complexes with some members of the nuclear receptor superfamily (see below). Sequence analysis has shown that HSP90 (the generic name for this family used here) is highly conserved among species (summarized in reference 15): human (HSP90), mouse (HSP84), chicken (HSP90), Drosophila (HSP83), Trypanosoma (HSP85), and yeast (HSP83) homologs display a minimum of 65% amino acid conservation.Members of the nuclear receptor superfamily display DNA-binding activity and translocate from the cytoplasm to the nucleus after acquiring a ligand. The inactive cytoplasmic forms of the glucocorticoid (18 [and references within], 35), estrogen (7 [and references within]), progesterone (6), and aryl hydrocarbon (AH) (29) receptors exist as stable complexes containing HSP90, whereas the ligand-activated nuclear forms lack HSP90. HSP90 is thought to negatively regulate activation and translocation of the cytoplasmic forms of the receptors in the absence of ligand (1). Substantial evidence suggests that binding of HSP90 may be essential to the subsequent activity of these receptors and that HSP90 may be involved in their initial folding. Glucocorticoid receptor translated from a reticulocyte lysate, which * Corresponding author.contains HSP90, displayed high-affinity hormone-binding activity, whe...

The cystic fibrosis transmembrane conductance regulator (CFTR) gene in man is controlled by a tightly regulated and weak promoter. The architecture of the CFTR promoter suggests regulatory characteristics that are consistent with the absence of a TATA-like sequence, including the ability to initiate RNA transcription at numerous positions. Detailed investigation of the most proximal region of the human CFTR gene promoter through deletion and mutational analysis reveals that expression is contingent on the conservation of the inverted CCAAT sequence. Basal expression of CFTR transcription and cAMP-mediated transcriptional regulation require the presence of an imperfect and inverted CCAAT element recognized as 5 -AATTGGAAGCAAAT-3 , located between 132 and 119 nucleotides upstream of the translational start site. RNA isolated from a transfected pancreatic cell line carrying integrated wild-type and mutant CFTR-directed transgenes was used to map the 5 termini of the transgenic transcripts. Analysis of the transcript termini by ribonuclease protection analysis reflects the direct association of the conserved inverted CCAAT sequence in promoting transcript initiation. Because of the requirement for the inverted CCAAT sequence for promoting transcription of CFTR, the involvement of CCAAT-binding factors is suspected in the regulation of CFTR gene transcription. To test this, we used electrophoretic mobility shift assays to demonstrate that the majority of the binding to the inverted CCAAT element, between ؊135 and ؊116, was easily competed for by binding to cognate nucleotide sequences for CCAAT-enhancer binding protein (C/EBP). An antibody specific for the C/EBP-related protein, C/EBP␦, detected C/EBP␦ as part of a nuclear protein complex bound to the inverted CCAAT sequence of the CFTR gene. Also, the detection of specific activating transcription factor/cyclic-AMP response element binding protein antigens by antibody supershift analysis of nuclear complexes suggest that species of this family of transcription factors could be involved in the formation of complexes with C/EBP␦ within the CFTR gene inverted CCAAT-like element. These studies raise the possibility of interactions between individual members of the C/EBP and activating transcription factor/cyclic-AMP response element binding protein families potentially contribute to the tight transcriptional control rendered by the CFTR gene promoter.The gene responsible for cystic fibrosis encodes the cystic fibrosis transmembrane conductance regulator (CFTR) 1 gene product (1, 2). The expression of CFTR is predominantly confined to specific epithelial cell types and is ordinarily expressed in low levels. The low levels and cell type-specific expression of CFTR appears to be dictated, primarily, by genomic sequences 5Ј upstream of the translational start of CFTR, which correspond to functional promoter sequences (3, 4). The requirements for active and cell type-specific CFTR transcription relies presumably within a narrow band of nucleotide sequences, proximal to the m...

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We have earlier prepared a pancreatic cancer-associated mucin, whose altered carbohydrate structure was recognized by Vicia villosa (VVA), Bauhinia purpurea (BPA), and peanut (PNA) lectins and which was found preferentially in the sera of patients with pancreatic or gastric cancer. Cancer-associated structures of the sugar chain on serum antigen may reflect those occurring in malignant tissues. Accordingly, we investigated the tissue distribution of carbohydrate structures reactive to these lectins by using lectin histochemistry in pancreatic cancer, gastric cancer, and colonic cancer tissue specimens and in their normal counterparts. VVA showed a higher affinity for pancreatic cancer (77.5%), gastric cancer (89%), and colonic cancer (87%) cells than for the cells of their normal counterparts, whose affinity was 0%, 41.7%, and 36.4%, respectively. PNA showed a higher affinity for pancreatic (70%) and colonic cancer cells (86.5%). BPA failed to show significant binding differences between neoplastic and normal cells in any of the pancreatic, gastric, or colonic tissue specimens. It did, however, bind to intraductal contents in most of the pancreatic cancer tissues but bound to intraductal contents in only a few chronic pancreatitis and normal pancreatic tissues. VVA and PNA did not bind to intraductal contents in any of the normal, chronic pancreatitis, or pancreatic cancer tissues. These results imply that, among the lectins used so far, VVA has the highest affinity for neoplastic cells, and it may provide a supplement for use in the pathologic diagnosis of malignant diseases.(ABSTRACT TRUNCATED AT 250 WORDS)