scholarly journals The tumor suppressor p53 is bound to RNA by a stable covalent linkage.

1991 ◽  
Vol 11 (3) ◽  
pp. 1598-1606 ◽  
Author(s):  
A Samad ◽  
R B Carroll
Keyword(s):  
Tumor Suppressor ◽  
Tryptic Peptide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Biologically Active ◽  
Intact Protein ◽  
Tumor Suppressor P53

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.

scholarly journals The tumor suppressor p53 is bound to RNA by a stable covalent linkage

1991 ◽  
Vol 11 (3) ◽  
pp. 1598-1606
Author(s):  
A Samad ◽  
R B Carroll
Keyword(s):  
Tumor Suppressor ◽  
Tryptic Peptide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Biologically Active ◽  
Intact Protein ◽  
Tumor Suppressor P53

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.

scholarly journals The tumor suppressor p53 and the oncoprotein simian virus 40 T antigen bind to overlapping domains on the MDM2 protein.

1993 ◽  
Vol 13 (11) ◽  
pp. 6849-6857 ◽  
Author(s):  
D R Brown ◽  
S Deb ◽  
R M Muñoz ◽  
M A Subler ◽  
S P Deb
Keyword(s):  
Amino Acids ◽  
Tumor Suppressor ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Domain ◽  
Tumor Suppressor P53 ◽  
Mdm2 Protein ◽  
N Terminus

The oncogene mdm2 has been found to be amplified in human sarcomas, and the gene product binds to the tumor suppressor p53. In this report, we describe the dissection of the MDM2-binding domain on p53 as well as the p53-binding domain on MDM2. We also demonstrate that the oncoprotein simian virus 40 T antigen binds to the product of cellular oncogene mdm2. We have constructed several N- and C-terminal deletion mutants of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding domain on MDM2 is between amino acids 1 and 58, while the C-terminal boundary is between amino acids 221 and 155. T antigen binds to an overlapping domain on the MDM2 protein. On the other hand, the MDM2-binding domain of p53 is defined by amino acids 1 and 159 at the N terminus. At the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the transactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibited the ability of human p53 to transactivate the promoter with p53-binding sites. Thus, human MDM2 protein, like the murine protein, can inactivate the transactivation ability of human p53. Interestingly, both the transactivation domain and the MDM2-binding domain of p53 are situated near the N terminus. We further show that deletion of the N-terminal 58 amino acids of MDM2, which eliminates p53 binding, also abolishes the capability of inactivating p53-mediated transactivation. This finding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.

The tumor suppressor p53 and the oncoprotein simian virus 40 T antigen bind to overlapping domains on the MDM2 protein

1993 ◽  
Vol 13 (11) ◽  
pp. 6849-6857
Author(s):  
D R Brown ◽  
S Deb ◽  
R M Muñoz ◽  
M A Subler ◽  
S P Deb
Keyword(s):  
Amino Acids ◽  
Tumor Suppressor ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Binding Domain ◽  
Tumor Suppressor P53 ◽  
Mdm2 Protein ◽  
N Terminus

The oncogene mdm2 has been found to be amplified in human sarcomas, and the gene product binds to the tumor suppressor p53. In this report, we describe the dissection of the MDM2-binding domain on p53 as well as the p53-binding domain on MDM2. We also demonstrate that the oncoprotein simian virus 40 T antigen binds to the product of cellular oncogene mdm2. We have constructed several N- and C-terminal deletion mutants of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding domain on MDM2 is between amino acids 1 and 58, while the C-terminal boundary is between amino acids 221 and 155. T antigen binds to an overlapping domain on the MDM2 protein. On the other hand, the MDM2-binding domain of p53 is defined by amino acids 1 and 159 at the N terminus. At the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the transactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibited the ability of human p53 to transactivate the promoter with p53-binding sites. Thus, human MDM2 protein, like the murine protein, can inactivate the transactivation ability of human p53. Interestingly, both the transactivation domain and the MDM2-binding domain of p53 are situated near the N terminus. We further show that deletion of the N-terminal 58 amino acids of MDM2, which eliminates p53 binding, also abolishes the capability of inactivating p53-mediated transactivation. This finding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.

scholarly journals Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts.

1994 ◽  
Vol 14 (7) ◽  
pp. 4398-4407 ◽  
Author(s):  
A S Alberts ◽  
M Montminy ◽  
S Shenolikar ◽  
J R Feramisco
Keyword(s):  
Transcriptional Activity ◽  
Expression Vector ◽  
Active Form ◽  
Simian Virus 40 ◽  
T Antigen ◽  
3T3 Cells ◽  
Phosphorylation State ◽  
Phosphorylated Creb ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We have examined the activity and phosphorylation state of the cyclic AMP (cAMP) response element binding factor (CREB) in intact NIH 3T3 cells following microinjection of expression plasmids encoding regulatory proteins of type 1 (PP1) and 2A (PP2A) serine/threonine-specific protein phosphatases. Changes in CREB phosphorylation in the injected cells were monitored by indirect immunofluorescence using an affinity-purified antiserum (Ab5322) which specifically recognizes CREB phosphorylated at Ser-133, and changes in transcriptional activity of CREB were monitored by expression of a reporter gene regulated by cAMP. cAMP-stimulated phosphorylation in NIH 3T3 cells is normally transient, and as expected, after stimulation of cells with cell-permeable cAMP analogs, the level of phosphorylated CREB was found to initially increase and then return to a basal level within 4 h. Microinjection of an expression vector encoding a constitutively active form of inhibitor 1 (I-1), a PP1-specific inhibitor, by itself resulted in an apparent increase in phosphorylated CREB in unstimulated cells. Moreover, injection of the I-1 vector resulted in the prolonged appearance of phosphorylated CREB in cells after cAMP stimulation. In contrast, injection of a plasmid encoding simian virus 40 small t antigen, which interacts with PP2A to inhibit its activity towards several phosphoprotein substrates, had no effect on the phosphorylation state of CREB in stimulated or unstimulated NIH 3T3 cells. Consistent with these results, injection of the I-1 expression vector activated expression from a coinjected CRE-lacZ reporter plasmid, indicating that the increased phosphorylation of CREB also activated its transcriptional activity. These results provide further evidence for a role of a PP1 as the primary protein (Ser/Thr) phosphatase regulating the dephosphorylation of Ser-133 and thereby limiting the transcriptional activity of CREB.

scholarly journals The interrelations between high- and low-molecular-weight forms of normal and mutant (Krabbe-disease) galactocerebrosidase

1980 ◽  
Vol 189 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Yoav Ben-Yoseph ◽  
Melinda Hungerford ◽  
Henry L. Nadler
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Krabbe Disease ◽  
Low Molecular Weight ◽  
Molecular Weight Form

Galactocerebrosidase (β-d-galactosyl-N-acylsphingosine galactohydrolase; EC 3.2.1.46) activity of brain and liver preparations from normal individuals and patients with Krabbe disease (globoid-cell leukodystrophy) have been separated by gel filtration into four different molecular-weight forms. The apparent mol.wts. were 760000±34000 and 121000±10000 for the high- and low-molecular-weight forms (peaks I and IV respectively) and 499000±22000 (mean±s.d.) and 256000±12000 for the intermediate forms (peaks II and III respectively). On examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the high- and low-molecular-weight forms revealed a single protein band with a similar mobility corresponding to a mol.wt. of about 125000. Antigenic identity was demonstrated between the various molecular-weight forms of the normal and the mutant galactocerebrosidases by using antisera against either the high- or the low-molecular-weight enzymes. The high-molecular-weight form of galactocerebrosidase was found to possess higher specific activity toward natural substrates when compared with the low-molecular-weight form. It is suggested that the high-molecular-weight enzyme is the active form in vivo and an aggregation process that proceeds from a monomer (mol.wt. approx. 125000) to a dimer (mol.wt. approx. 250000) and from the dimer to either a tetramer (mol.wt. approx. 500000) or a hexamer (mol.wt. approx. 750000) takes place in normal as well as in Krabbe-disease tissues.

Transport and proteolytic processing of rabbit cardiac cathepsin D

1985 ◽  
Vol 248 (1) ◽  
pp. C135-C144
Author(s):  
A. M. Samarel ◽  
A. G. Ferguson ◽  
S. W. Worobec ◽  
M. Lesch
Keyword(s):  
Gel Electrophoresis ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Proteolytic Processing ◽  
Time Dependent ◽  
Low Density ◽  
Enzyme Maturation

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.

scholarly journals Purification and structural characterization of a cartilage matrix protein

1981 ◽  
Vol 197 (2) ◽  
pp. 367-375 ◽  
Author(s):  
M Paulsson ◽  
D Heinegård
Keyword(s):  
Molecular Weight ◽  
Matrix Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cartilage Matrix ◽  
Sedimentation Equilibrium ◽  
Intact Protein ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient

The cartilage matrix protein is a major non-collagenous protein in bovine cartilage. It was purified from a 5 M-guanidinium chloride extract of bovine tracheal cartilage by sequential CsCl-density-gradient centrifugation, gel chromatography in guanidinium chloride and differential precipitation. The molecular weight of the intact protein is 148 000, determined by sedimentation-equilibrium centrifugation. It was dissociated to three subunits of molecular weight 52 000 by reduction of disulphide bonds. The cartilage matrix protein was insoluble in low-salt solutions and behaved abnormally on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The content of cysteine was high, whereas the contents of aromatic amino acids were low. The carbohydrate content was 3.9% (w/w). Glycopeptides obtained after papain digestion were heterogenous on gel chromatography. Asparagine/aspartic acid was enriched in the purified glycopeptides, indicating the presence of N-glycosidic linkages to protein.

scholarly journals Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor.

1988 ◽  
Vol 8 (3) ◽  
pp. 1011-1018 ◽  
Author(s):  
M K Sauer ◽  
D J Donoghue
Keyword(s):  
Biological Activity ◽  
Growth Factor ◽  
Disulfide Bonds ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Biologically Active ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Type V

The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond.

scholarly journals Simian Virus 40 Large T Antigen's Association with the CUL7 SCF Complex Contributes to Cellular Transformation

2005 ◽  
Vol 79 (18) ◽  
pp. 11685-11692 ◽  
Author(s):  
Jocelyn S. Kasper ◽  
Hiroshi Kuwabara ◽  
Takehiro Arai ◽  
Syed Hamid Ali ◽  
James A. DeCaprio
Keyword(s):  
Tumor Suppressor ◽  
Mouse Embryo ◽  
Family Members ◽  
Simian Virus 40 ◽  
Cellular Transformation ◽  
Simian Virus ◽  
T Antigen ◽  
Mouse Embryo Fibroblasts ◽  
Embryo Fibroblasts ◽  
F Box Protein

ABSTRACT Simian virus 40 large T antigen (T Ag) is capable of immortalizing and transforming rodent cells. The transforming activity of T Ag is due in large part to perturbation of the tumor suppressor proteins p53 and the retinoblastoma (pRB) family members. Inactivation of these tumor suppressors may not be sufficient for T Ag-mediated cellular transformation. It has been shown that T Ag associates with an SCF-like complex that contains a member of the cullin family of E3 ubiquitin ligases, CUL7, as well as SKP1, RBX1, and an F-box protein, FBXW8. We identified T Ag residues 69 to 83 as required for T Ag binding to the CUL7 complex. We demonstrate that Δ69-83 T Ag, while it lost its ability to associate with CUL7, retained binding to p53 and pRB family members. In the presence of CUL7, wild-type (WT) T Ag but not Δ69-83 T Ag was able to induce proliferation of mouse embryo fibroblasts, an indication of cellular transformation. In contrast, WT and Δ69-83 T Ag enabled mouse embryo fibroblasts to proliferate to similarly high densities in the absence of CUL7. Our data suggest that, in addition to p53 and the pRB family members, T Ag serves to bind to and inactivate the growth-suppressing properties of CUL7. In addition, these results imply that, at least in the presence of T Ag, CUL7 may function as a tumor suppressor.

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