Induction Of Response Genes Research Articles

CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases.

The mechanism by which Ca2+ mediates gene induction in response to membrane depolarization was investigated. The adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) was shown to function as a Ca(2+)-regulated transcription factor and as a substrate for depolarization-activated Ca(2+)-calmodulin-dependent protein kinases (CaM kinases) I and II. CREB residue Ser133 was the major site of phosphorylation by the CaM kinases in vitro and of phosphorylation after membrane depolarization in vivo. Mutation of Ser133 impaired the ability of CREB to respond to Ca2+. These results suggest that CaM kinases may transduce electrical signals to the nucleus and that CREB functions to integrate Ca2+ and cAMP signals.

Heat shock factor and the heat shock response

Peter K. Sorger Department of Microbiology and Immunology University of California San Francisco, California 94143-0502 The induction of eukaryotic heat shock genes in response to a temperature upshift is mediated by the binding of a transcriptional activator, heat shock factor, to a short highly conserved DNA sequence known as the heat shock element. This review will discuss progress in several or- ganisms in characterizing heat shock factor and will sug- gest preliminary answers to the following questions. How is the DNA-binding activity and transcriptional potency of heat shock factor regulated? How is autoregulation of heat shock protein (hsp) synthesis achieved? What is the mech- anism by which heat shock is sensed? Heat Shock Elements and Their Interaction with Heat Shock Factor Heat shock elements are best described as contiguous arrays of variable numbers of the 5 bp sequence nGAAn arranged in alternating orientation (n denotes less strongly conserved nucleotides that nevertheless may be involved in important DNA-protein interactions; Figure 1; Xiao and Lis, 1988; Amin et al., 1988). At least two nGAAn units are needed for high affinity binding of heat shock factor in vitro, and these may be arranged either head-to-head (nGAAnnTTCn) or tail-to-tail (nTTCnnGAAn; Perisic et al., 1989). How can heat shock factor bind to these structurally distinct sites as well as to heat shock elements with larger numbers of 5 bp units? The answer may lie in the oligo- merit nature of the heat shock factor protein (Figure 2). Heat shock factor from both Saccharomyces cerevisiae (SC-HSF) and Drosophila (D-HSF) associates to form pro- tein trimers in solution and when bound to DNA (Perisic et al., 1989; Sorger and Nelson, 1989). It is not clear, how- ever, whether heat shock factor exists in vivo primarily as a trimeric, hexameric, or possibly even larger complex. Each subunit of a D-HSF multimer is thought to bind to a single nGAAn unit, and the binding to successive units is highly cooperative (Xiao et al., 1991). The coiled-coil structure that has been proposed to form the interface between heat shock factor monomers is inherently three- fold symmetric (see below). Thus, the binding of heat shock factor trimers to DNA (which is inherently two-fold symmetric) would require a flexible hinge between the tri- merization and DNA-binding surfaces of individual mono- mers. Flexibility at this hinge may also be exploited in the binding of subunits to differently oriented nGAAn units. The binding of trimers to adjacent sites is also highly cooperative (Shuey and Parker, 1986): in vitro, the dissoci- ation rate from DNA of a complex of two DNA-bound tri- mers is more than three orders of magnitude lower than that of a single trimer (Xiao et al., 1991). Thus, even when the molarity of sites exceeds that of the heat shock factor protein, heat shock factor preferentially forms large com-

Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor‐induced signals for gene activation

The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF)-stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors.

Strain-Specific Determination of the Degree of Thyroid Cell MHC Class II Antigen Expression: Evaluation of Established Wistar and Fisher Rat Thyroid Cell Lines*

The major histocompatibility complex (MHC) genes, primarily of the MHC class II region, are linked to increased susceptibility to autoimmune thyroid disease in animals and humans. The quantity of MHC class II antigens that are expressed on the appropriate cell surface, as well as their allotype, are of vital importance to immune function. We have, therefore, compared MHC class II (RT1.D) gene activation in a newly available Wistar rat thyroid (WRT) cell line (a thyroiditis-susceptible strain) with a cloned cell (1B-6) derived from the Fisher rat thyroid cell line (FRTL-5) (a thyroiditis-resistant strain) to determine differences in their degree of MHC class II gene activation and antigen expression. There was no detectable constitutive MHC class II antigen or RT1.D alpha-chain messenger RNA expression in either WRT or 1B-6 cells. Cultures of both cells expressed MHC class II gene induction in response to recombinant rat gamma-interferon (gamma IF). After 72 h of exposure to 25 U/ml gamma IF, over 80% of WRT cells expressed class II antigen, detected by flow cytometric assessment, as compared to only 15% of 1B-6 cells. This earlier and greater expression of MHC class II antigen was reflected in the RT1.D alpha-chain mRNA responses with peak levels in WRT cells after only 24 h of exposure to gamma IF, a period in which 1B-6 cells showed only minor increases in mRNA. To examine whether these differences in class II MHC expression were thyroid-cell specific, primary pulmonary fibroblast lines from Wistar and Fisher rats were developed and a similar, although less marked, variation in susceptibility to gamma IF was observed. Hence, those rats strains that are resistant to autoimmune thyroid disease exhibit a fundamental difference in their MHC class II gene responsiveness to cytokines, which may contribute to their disease susceptibility profiles.

GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast.

The yeast GCN4 gene product is necessary for the transcriptional induction of many amino acid biosynthetic genes in response to conditions of amino acid starvation. We synthesized radioactively pure GCN4 protein by in vitro translation of mRNA produced by in vitro transcription with SP6 RNA polymerase. GCN4 protein binds specifically to the 20 bp region of the HIS3 gene that is critical for transcriptional regulation in vivo and contains the TGACTC sequence common to coregulated genes. A synthetic GCN4 mutant protein lacking the 40 C-terminal amino acids fails to bind DNA; this correlates with a gcn4 mutant gene that is nonfunctional in vivo. Finally, GCN4 protein binds to the promoter regions of coordinately regulated genes, but not to analogous regions of other genes. We suggest that GCN4 protein is a specific transcription factor, and we describe a molecular model for the general control of amino acid biosynthetic genes.

The regulation and function of small heat‐shock protein synthesis

AbstractThe four small heat shock protein genes of Drosophila are tightly linked at the level of DNA, and are coordinately regulated. In cultured cell lines their expression is induced by high temprature shock and by physiological doses of ecdysterone. In vivo, small heat shock gene expression is developmentally regulated. Using recombinant DNA clones we have characterized and compared small hsp gene induction in response to the two independent stimuli.