Showing papers by "Michael G. Rosenfeld published in 1987"

Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor.

Abstract: The epidermal growth factor (EGF) receptor is a transmembrane glycoprotein of relative molecular mass 170,000 with intrinsic ligand-dependent protein tyrosine kinase activity1–6. Binding of EGF to its receptor activates a number of immediate biochemical processes, such as alterations of intracellular free calcium, pH, and increased transcription of several responsive genes, which usually culminate many hours later in DNA replication and cell division7–14. Abolishing the tyrosine kinase activity of three related oncogenes, v-src, v-mos, and v-fps, eliminates their capacity to transform cells15–17. Several reports have suggested that specific aspects of EGF receptor function are independent of the intrinsic tyrosine kinase activity18,19; however, these studies used an antibody against EGF receptor which failed to activate phosphoryla-tion of exogenous substrates18 and an insertional mutation in the EGF receptor tyrosine kinase domain which had not been shown to abolish protein kinase activity in cells19. Because many transmembrane receptors interact with intrinsic membrane proteins to activate second messenger systems, it is important to resolve experimentally whether mechanisms, in addition to activation of the intrinsic tyrosine kinase activity, mediate some EGF actions. From functional analyses of an EGF receptor containing a single amino-acid mutation at a site required for phosphate transfer from ATP, we conclude that the tyrosine kinase activity of the EGF receptor is essential for the diverse biochemical effects of EGF, including rapid alterations in intracellular calcium, activation of gene transcription, receptor down-regulation and the ultimate stimulatory effects on cell proliferation.

A c- erb-A binding site in rat growth hormone gene mediates trans -activation by thyroid hormone

Abstract: The substance 3,5,3-triiodothyronine (T3) stimulates growth hormone gene transcription in rat pituitary tumour cells1–4. This stimulation is thought to be mediated by the binding of nuclear T3 receptors to regulatory elements 5' to the transcriptional start site5–8. Understanding of the mechanism by which thyroid hormone activates gene transcription has been limited by failure to purify nuclear T3 receptors because of their low abundance, and by the absence of defined T3 receptor-DNA binding sites affecting T3 regulation. Recently, human and avian c-erb-A gene products have been shown to bind thyroid hormone with high affinity9,10 and to have a molecular weight and nuclear association characteristic of the thyroid hormone receptor. In the present report, we describe the development of an avidin–biotin complex DNA-binding assay which can detect specific, high-affinity binding of rat pituitary cell T3 receptors to the sequence 5'CAGGGACGTGACCGCA3', located 164 base pairs 5' to the transcriptional start site of the rat growth hormone gene. An oligonucleotide containing this sequence transferred T3 regulation to the herpes simplex virus thymidine kinase promoter in transfected rat pituitary GC2 cells, and specifically bound an in vitro translation product of the human placental c-erb-A gene. The data provide supporting evidence that the human c-erb-A gene product mediates the transcriptional effects of T3 and also that GC2 cell nuclear extracts contain additional factors that modify the binding of pituitary T3 receptors to the rat growth hormone gene T3 response element.

Molecular cloning of a brain-specific calcium/calmodulin-dependent protein kinase.

Abstract: A calcium/calmodulin-dependent protein kinase type II (CaM-K) alpha-subunit cDNA has been cloned from rat brain. This enzyme is encoded by a 5.1-kilobase mRNA expressed exclusively in the brain. Hybridization histochemistry reveals that the CaM-K mRNA expression corresponds to the distribution of the immunoreactive alpha-subunit protein, suggesting that the high enzyme levels in specific brain areas reflect regional differences in gene expression. The sequence of CaM-K alpha-subunit cDNA indicates a 478-amino acid (54-kDa) protein with three functional domains. The domain organization suggests a structural model for calcium/calmodulin-dependent and independent states that might subserve short- and long-term responses to transient stimuli.

Transcriptional control mechanisms

Abstract: This book consists of papers presented at a symposium on mechanisms of transcriptional control Topics discussed include: 5'-regulatory elements of the human HPRT gene;nuclear factors interacting with immunoglobulin heavy and K enhancers;mitochondinal transcription factor binds novel control elements of both major promoters of human mt DNA;RNA processing and translation;chromatin structure and assembly;hormonal regulation of transcription and eukaryotic transcription systems

Developmental and hormonal regulation of neuroendocrine gene transcription.

Abstract: Publisher Summary The quantitative regulation of gene transcription during development and by hormones appears to require the interaction of specific rate-limiting transcriptional factors that bind to structurally distinct genomic sequences. The precise molecular mechanisms by which the binding of these trans-acting factors increases the rate of accurate transcriptional initiation remain unknown; however, it is clear that the regulatory cis-active regions can represent either simple or complex elements. In the case of heritable patterns of neuroendocrine expression, a combinatorial reaction of several sequences and their cognate transcription factors appears requisite for cell-specific gene transcription. The multi-factorial nature of this regulation provides the possibility for a more restricted pattern of gene expression than that exhibited by the cognate trans-acting transcription factors. In the case of the rat prolactin gene, three to five discrete factors may be required for the upstream enhancer function. Additional important cell-specific enhancers appear to be present in proximity to the promoter. By contrast, several regulatory cis-active elements, such as those that transfer hormone regulation, appear to bind a unique transcription factor, which may be sufficient for activation of gene transcription.

Developmental control of a calcium-regulating neuroendocrine gene

Abstract: Publisher Summary This chapter focuses on alternative RNA processing events operate in a tissue-specific, developmentally regulated fashion in the neuroendocrine system to produce alternative, discrete polypeptide products serving to further increase the diversity required to subserve the biological demands of complex organisms. The similarity of the alternative RNA processing events in genes of the neuroendocrine system with those of other families of eukaryotic genes suggest that common underlying biochemical mechanisms may operate in this form of regulated expression of many eukaryotic transcription units. Using an antisera generated against a synthetic peptide corresponding to the 14 C1-terminal amino acids of Calcitonin Gene-Related Product (CGRP), immunoreactive CGRP was identified in a unique distribution in a large number of cell groups and pathways in the central nervous system distinct from that of any known neuropeptide. The possible existence of other gene products related to CGRP was investigated by screening libraries of chimaeric plasmids containing inserts complementary to mRNAs from rat medullary thyroid carcinomas with a clonal alpha-CGRP cDNA probe. Sequence- or site-specific poly(A) site selection, or choice of splice acceptor were considered as the alternative regulated event that accounts for the developmentally-regulated RNA processing events resulting in CGRP production.