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Biomedical subjects

R A Weinberg

Publications and source records attributed to R A Weinberg.

At least 19 recordsLinked to original sources
PubMed1995

Requirement for TFIIH kinase activity in transcription by RNA polymerase II.

An array of tandem heptapeptide repeats at the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II constitute a highly conserved structure essential for viability. Studies have established that the CTD is phosphorylated at different stages of the transcription cycle, and that it may be involved in transcriptional regulation. The exact role of the CTD remains elusive, as in vitro reconstituted transcription using the adenovirus major late promoter does not require the CTD. Previous studies showed that transcription from the murine dihydrofolate reductase (DHFR) promoter can be only accomplished by the form of RNA polymerase II that contains the hypophosphorylated CTD (RNAPIIA), but not by the form that lacks it (RNAPIIB). Here we show that the CTD, but not its phosphorylation, is required for initiation of transcription. We also show that transcription requires CTD kinase activity provided by the CDK subunit of TFIIH.

Adenoviridae↗
PubMed1995

Cyclin D1 provides a link between development and oncogenesis in the retina and breast.

Mice lacking cyclin D1 have been generated by gene targeting in embryonic stem cells. Cyclin D1-deficient animals develop to term but show reduced body size, reduced viability, and symptoms of neurological impairment. Their retinas display a striking reduction in cell number due to proliferative failure during embryonic development. In situ hybridization studies of normal mouse embryos revealed an extremely high level of cyclin D1 in the retina, suggesting a special dependence of this tissue on cyclin D1. In adult mutant females, the breast epithelial compartment fails to undergo the massive proliferative changes associated with pregnancy despite normal levels of ovarian steroid hormones. Thus, steroid-induced proliferation of mammary epithelium during pregnancy may be driven through cyclin D1.

Animals↗
PubMed1995

Cooperative binding of transforming growth factor (TGF)-beta 2 to the types I and II TGF-beta receptors.

TGF-beta 1 binds with high affinity (KD = 25-50 pM) directly to the TGF-beta type II receptor serine-threonine kinase (T beta-RII) in the absence of expression of the TGF-beta type I or III receptors (T beta-RI and T beta-RIII). The serine-threonine kinase T beta-RI is essential for TGF-beta 1 signaling but not for binding to T beta-RII. TGF-beta 2, in contrast, does not bind directly to T beta-RII, although coexpression of T beta-RIII does allow binding and cross-linking of TGF-beta 2 to T beta-RII. Here we show that in transfected COS cells binding and cross-linking of 125I-TGF-beta 2 to T beta-RI or T beta-RII requires expression of both receptors. In cells transfected with the c-myc-tagged human T beta-RII cDNA, only low amounts of 125I-TGF-beta 2 cross-linked to T beta-RI and T beta-RII were detected even with high concentrations (700 pM) of ligand. Cotransfection of the influenza-hemagglutinin-tagged human T beta-RI cDNA dramatically increased the binding of TGF-beta 2 to T beta-RII; the concentration of 125I-TGF-beta 2 required for half-maximal binding and cross-linking to T beta-RI and T beta-RII was approximately 40 pM. Coimmunoprecipitation studies showed that the high affinity receptor for TGF-beta 2 is composed of a hetero-oligomer of T beta-RI and T beta-RII. Thus TGF-beta 1 and -beta 2 bind to TGF-beta receptors in different ways; TGF-beta 1 binds directly to T beta-RII, while binding of TGF-beta 2 to T beta-RII requires coexpression of T beta-RI or T beta-RIII.

Activin Receptors, Type I↗
PubMed1995

Growth suppression by p16ink4 requires functional retinoblastoma protein.

p16ink4 has been implicated as a tumor suppressor that is lost from a variety of human tumors and human cell lines. p16ink4 specifically binds and inhibits the cyclin-dependent kinases 4 and 6. In vitro, these kinases can phosphorylate the product of the retinoblastoma tumor suppressor gene. Thus, p16ink4 could exert its function as tumor suppressor through inhibition of phosphorylation and functional inactivation of the retinoblastoma protein. Here we show that overexpression of p16ink4 in certain cell types will lead to an arrest in the G1 phase of the cell cycle. In addition, we show that p16ink4 can only suppress the growth of human cells that contain functional pRB. Moreover, we have compared the effect of p16ink4 expression on embryo fibroblasts from wild-type and RB homozygous mutant mice. Wild-type embryo fibroblasts are inhibited by p16ink4, whereas the RB nullizygous fibroblasts are not. These data not only show that the presence of pRB is crucial for growth suppression by p16ink4 but also indicate that the pRB is the critical target acted upon by cyclin D-dependent kinases in the G1 phase of the cell cycle.

Animals↗
PubMed1995

Suppression of intestinal neoplasia by DNA hypomethylation.

We have used a combination of genetics and pharmacology to assess the effects of reduced DNA methyltransferase activity on ApcMin-induced intestinal neoplasia in mice. A reduction in the DNA methyltransferase activity in Min mice due to heterozygosity of the DNA methyltransferase gene, in conjunction with a weekly dose of the DNA methyltransferase inhibitor 5-aza-deoxycytidine, reduced the average number of intestinal adenomas from 113 in the control mice to only 2 polyps in the treated heterozygotes. Hence, DNA methyltransferase activity contributes substantially to tumor development in this mouse model of intestinal neoplasia. Our results argue against an oncogenic effect of DNA hypomethylation. Moreover, they are consistent with a role for DNA methyltransferase in the generation of the C to T transitions seen at high frequency in human colorectal tumors.

Adenomatous Polyposis Coli↗
PubMed1995

Genetic studies reveal that myristoylCoA:protein N-myristoyltransferase is an essential enzyme in Candida albicans.

MyristoylCoA:protein N-myristoyltransferase (Nmt) catalyses the co-translational, covalent attachment of myristate (C14:0) to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction. The NMT1 gene is essential for vegetative growth of Saccharomyces cerevisiae. Studies were carried out to determine if Nmt is also essential for vegetative growth of the pathogenic fungus Candida albicans. A strain of C. albicans was constructed in which one copy of NMT was partially deleted and disrupted. A Gly-447-->Asp mutation was introduced into the second NMT allele. This mutation produced marked reductions in catalytic efficiency at 24 and 37 degrees C, as judged by in vitro kinetic studies of the wild-type and mutant enzymes which had been expressed in, and purified from, Escherichia coli. The growth characteristics of isogenic NMT/NMT, NMT/delta nmt, and nmt delta/nmtG447D C. albicans strains were assessed under a variety of conditions. Only the nmt delta/nmtG447D strain required myristate for growth. This was true at both 24 and 37 degrees C. Palmitate could not substitute for myristate. Incubation of nmt delta/nmtG447D cells at 37 degrees C in the absence of myristate resulted in cell death as observed by the inability to form colonies on media supplemented with 500 microM myristate. Studies in an immunosuppressed-mouse model of C. albicans infection revealed that the NMT/delta nmt strain produced 100% lethality within 7 d after intravenous administration while the isogenic nmt delta/nmtG447G strain produced no deaths even after 21 d. These observations establish that Nmt is essential for vegetative growth of C. albicans and suggest that inhibitors of this acyltransferase may be therapeutically useful fungicidal agents.

Acyltransferases↗
PubMed1992

Effects of an Rb mutation in the mouse.

The retinoblastoma gene is mutated in several types of human cancer and is the best characterized of the tumour-suppressor genes. A mouse strain has been constructed in which one allele of Rb is disrupted. These heterozygous animals are not predisposed to retinoblastoma, but some display pituitary tumours arising from cells in which the wild-type Rb allele is absent. Embryos homozygous for the mutation die between days 14 and 15 of gestation, exhibiting neuronal cell death and defective erythropoiesis.

Amino Acid Sequence↗
PubMed1992

Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.

Cell Nucleus↗
PubMed1992

The integration of molecular genetics into cancer management.

Many of the phenotypes of cancer cells and tumors can now be traced to specific mutations in the genomes of these cells. These mutations may activate oncogenes, providing mitogenic stimulus to these cells. Alternatively, they may inactivate tumor-suppressor genes, relieving growth-inhibiting constraints placed on these cells. These genetic lesions together provide many of the explanations for the deregulated growth of tumor cells.

Cell Division↗
PubMed1992

Association between GTPase activators for Rho and Ras families.

The ras-related low-molecular-mass GTPases participate in signal transduction involving a variety of cellular functions, including cell-cycle progression, cellular differentiation, cytoskeletal organization, protein transport and secretion. The cycling of these proteins between GTP-bound and GDP-bound states is partially controlled by GTPase activating proteins (GAPs) which stimulate the intrinsic GTP-hydrolysing activity of specific GTPases. The ras GTPase-activating protein (Ras-GAP) forms a complex with a second protein, p190 (M(r) 190,000), in growth-factor stimulated and tyrosine-kinase transformed cells. At its carboxy-terminal end, p190 contains a region that is conserved in the breakpoint cluster region, n-chimaerin, and Rho-GAP. Each of these three proteins exhibits GAP activity for at least one member of the rho family of small GTPases. We have tested recombinant p190 protein for GAP activity on GTPases of the ras, rho and rab families, and show here that p190 can function as a GAP specifically for members of the rho family. Consequently, the formation of a complex between Ras-GAP and p190 in growth-factor stimulated cells may allow the coupling of signalling pathways that involve ras and rho GTPases.

Animals↗
PubMed1992

Molecular cloning of cDNAs encoding the GAP-associated protein p190: implications for a signaling pathway from ras to the nucleus.

In mitogenically stimulated and tyrosine kinase-transformed cells, a substantial fraction of the ras GTPase-activating protein (GAP) forms a complex with a protein termed p190. We have cloned several cDNAs encoding the p190 protein. Analysis of the predicted protein sequence reveals three distinct domains with homology to previously described sequences. An N-terminal domain of p190 contains sequence motifs that are found in all of the known GTPases. At the C-terminus of the protein is a domain that contains sequences very similar to those found in the breakpoint cluster region gene product, n-chimerin, and rho GAP, all of which have been shown to possess intrinsic GAP activity on small GTPases. Finally, a 778 aa segment in the middle of p190 is nearly identical in sequence to a recently described transcriptional repressor. This raises the possibility that p190, acting via GAP, can transduce signals from p21ras to the nucleus, perhaps affecting expression of specific cellular genes.

Amino Acid Sequence↗
PubMed1992

An interaction between p21ras and heat shock protein hsp60, a chaperonin.

Ras proteins play a crucial role in the development of neoplasia and in signal transduction in normal cells. In a search for proteins interacting with p21ras, we previously identified a protein of 60 kDa (p60) through use of a chemical cross-linker. Using information from partial amino acid sequencing of the purified protein, we isolated full-length cDNA clones encoding this 60-kDa protein. Nucleotide sequence analysis revealed that p60 is the murine heat shock protein hsp60, a chaperonin. Association of hsp60 with p21ras appears physiological, as the amount of hsp60 complexed to p21ras was similar even in cells over-expressing p21ras, and reversing the order of cross-linking and lysis of the cells, which releases large amounts of hsp60 from mitochondria, did not alter the amount of hsp60 cross-linked to p21ras.

Amino Acid Sequence↗
PubMed1992

Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase.

A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1. Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.

Amino Acid Sequence↗
PubMed1992

The retinoblastoma protein and the regulation of cell cycling.

Increasing attention has been focused on how the retinoblastoma (RB) protein regulates cell growth. Recent evidence indicates that it is a substrate for phosphorylation by cyclin-dependent kinase-cyclin complexes and suggests that this phosphorylation modulates the ability of this protein to regulate transit through the cell cycle, perhaps in its G1 phase.

Animals↗
PubMed1992

Negative regulators of growth.

The proliferation of cells is regulated by countervailing positively- and negatively-acting signaling networks. The anti-proliferative signals, the study of which has been much neglected until recently, are often conveyed by growth-inhibitory peptides. Elements that mediate the cellular response to growth inhibitors are encoded by tumor suppressor genes that if lost may lead to the runaway growth of the cancer cell.

Genes, Tumor Suppressor↗