Biomedical subjects

L J Ransone

Publications and source records attributed to L J Ransone.

26 records · Page 2Linked to original sources

PubMed1989

Fos-Jun interaction: mutational analysis of the leucine zipper domain of both proteins.

Jun and Fos oncoproteins form a complex that regulates transcription from promoters containing AP-1 binding sites. The 'leucine zipper' domain of both Fos and Jun is necessary for the formation of the heterodimer, but the role of specific leucine residues is unclear. We have used site-specific mutagenesis to examine the contribution of individual leucine residues to the formation of a stable Fos-Jun protein complex and the binding of this complex to the AP-1 site. Mutation of a single leucine in either Fos or Jun had no effect on protein complex formation. Furthermore, mutations of two consecutive leucines in Jun did not interfere with heterodimer formation; however, in the case of Fos, two consecutive mutations resulted in an inability to form a heterodimer. Although mutagenesis of the first leucine of the heptad repeat had no effect on protein complex formation, this mutation in either Fos or Jun drastically reduced the affinity of the complex for DNA. Thus, both Fos and Jun contribute directly to the DNA-binding potential of the heterodimer.

Amino Acid Sequence↗

PubMed1989

Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: evidence for phosphorylation.

Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.

Amino Acids↗

PubMed1989

fos and jun interaction: the role of the leucine zipper.

Jun and fos oncoproteins form a complex which regulates transcription from promoters containing AP-I binding sites. The "leucine zipper" domain of both fos and jun is necessary for the formation of the heterodimer, but the role of specific leucine residues is unclear. We have used site-specific mutagenesis to examine the contribution of individual leucine residues to the formation of a stable fos/jun protein complex and the binding of this complex to the AP-I site. Mutation of a single leucine in either fos or jun had no effect on protein complex formation. Furthermore, mutations of two consecutive leucines in jun did not interfere with heterodimer formation; however, in the case of fos, two consecutive mutations resulted in an inability to form a heterodimer. Although mutagenesis of the first leucine of the heptad repeat had no effect on protein complex formation, this mutation in either fos or jun drastically reduced the affinity of the complex for DNA. Thus, both fos and jun contribute directly to the DNA binding potential of the heterodimer.

Amino Acid Sequence↗

PubMed1989

fos-jun Conspiracy: implications for the cell.

Two nuclear oncoproteins, fos and jun (AP-1), cooperate in forming a very stable heterodimeric complex that binds to the AP-1 site with increased affinity. The 'leucine zipper' domain of both fos and jun is necessary for the formation of this heterodimer. Mutations of single residues within the leucine zipper domain had no effect on protein complex formation. However, mutagenesis of the first leucine of the heptad repeat in either fos or jun basic regions and alteration of the spacing between the basic and leucine zipper domains indicate that the basic region of fos has a crucial role in determining the DNA binding affinity of the transcriptional complex. Mutations of the basic amino acids in fos protein prevent binding to TPA (phorbol ester)-responsive element (TRE) in the presence of wild-type jun protein. Thus fos protein appears to be dominant in jun-fos binding to DNA, even though fos alone cannot bind to TRE. Mutants in the basic regions of fos and jun can be exploited as dominant-negative mutants to ablate their normal cellular function.

Amino Acid Sequence↗

PubMed1988

Direct interaction between fos and jun nuclear oncoproteins: role of the 'leucine zipper' domain.

Gene expression is modulated by the specific interactions of nuclear proteins with unique regulatory sequences in the genome. Proteins involved in transcriptional regulation seem to be either transcription factors or transcription modulators and their interactions are crucial in determining whether the expression of a specific gene is activated or repressed. Recently, the product of the proto-oncogene jun has been identified as the transcription factor AP-1, whereas nuclear oncoproteins fos and myc have been implicated in transcriptional transregulation of several promoters. Furthermore, the products of the fos and jun proto-oncogenes are associated in some transcription complexes. Although the nature of the association is unclear, the two proteins co-immunoprecipitate with fos antibodies in nuclear extracts. Here, we report studies that demonstrate that the fos protein directly modulates jun function by means of a heterodimer of fos and jun proteins. The fos 'leucine zipper' domain is necessary for the DNA binding of the heterodimer; a distinct domain, localized in the C-terminal region of the fos protein, is responsible for transcriptional regulation.

DNA↗

PubMed1988

A heat-sensitive inhibitor in poliovirus-infected cells which selectively blocks phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase.

We partially purified an inhibitor from poliovirus-infected HeLa cells which specifically blocked phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the double-stranded RNA-activated protein kinase. The inhibitory activity eluted from a sizing column with an approximate molecular weight of 80,000 to 100,000 and was sensitive to heat, suggesting a protein nature for the inhibitor. No specific virus-encoded protein purified with the inhibitor. The inhibition of phosphorylation of the alpha subunit of eucaryotic initiation factor 2 was not due to a protein phosphatase associated with the inhibitor. The inhibitor did not seem to prevent phosphorylation of the double-stranded RNA-activated protein kinase but inhibited the phosphorylation of the alpha subunit of eucaryotic initiation factor 2 by the activated kinase. Double-stranded RNA-induced inhibition of in vitro protein synthesis in reticulocyte lysates could be prevented by the addition of the partially purified inhibitor during preincubation of lysate with double-stranded RNA.

Autoradiography↗

PubMed1987

Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase.

Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.

Electrophoresis, Agar Gel↗

PubMed1987

Activation of double-stranded RNA-activated protein kinase in HeLa cells after poliovirus infection does not result in increased phosphorylation of eucaryotic initiation factor-2.

Protein kinase activity in general is stimulated at least 5- to 10-fold in ribosomal salt wash preparations from poliovirus-infected HeLa cells compared with those from mock-infected cells. The stimulation of kinase activity is manifested by increased phosphorylation of ribosome-associated polypeptides having approximate molecular weights of 135,000, 120,000, 85,000, 68,000, 65,000, 40,000, 28,000, 25,000, and 21,000. The Mr 68,000 phosphoprotein is structurally identical to the interferon-induced, double-stranded RNA-activated protein kinase (P1) which phosphorylates the alpha subunit of eucaryotic initiation factor-2 (eIF-2). A similar protein of Mr 68,000 is more phosphorylated in poliovirus-infected cells than in mock-infected cells. Increased phosphorylation of P1 protein in poliovirus-infected cells, however, does not result in an increased phosphorylation of the alpha subunit of endogenous or exogenously added eIF-2, both in vitro and in vivo. These results suggest that a mechanism must exist in poliovirus-infected HeLa cells which prevents further phosphorylation of eIF-2 by the activated kinase.

Enzyme Activation↗