scholarly journals Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

1988 ◽  
Vol 8 (8) ◽  
pp. 3235-3243 ◽  
Author(s):  
L A Feig ◽  
G M Cooper
Keyword(s):  
Cell Proliferation ◽  
Inhibitory Effect ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Protein ◽  
Mammalian Expression ◽  
Guanine Nucleotide Binding ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP

1988 ◽  
Vol 8 (8) ◽  
pp. 3235-3243 ◽  
Author(s):  
L A Feig ◽  
G M Cooper
Keyword(s):  
Cell Proliferation ◽  
Inhibitory Effect ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Protein ◽  
Mammalian Expression ◽  
Guanine Nucleotide Binding ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

scholarly journals A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

1986 ◽  
Vol 6 (5) ◽  
pp. 1843-1846 ◽  
Author(s):  
A G Papageorge ◽  
B M Willumsen ◽  
M Johnsen ◽  
H F Kung ◽  
D W Stacey ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
S Phase ◽  
3T3 Cells ◽  
Ras Gene ◽  
Mutant Proteins ◽  
Ras Protein ◽  
Ras Genes ◽  
Essential Function ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene

1986 ◽  
Vol 6 (5) ◽  
pp. 1843-1846
Author(s):  
A G Papageorge ◽  
B M Willumsen ◽  
M Johnsen ◽  
H F Kung ◽  
D W Stacey ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
S Phase ◽  
3T3 Cells ◽  
Ras Gene ◽  
Mutant Proteins ◽  
Ras Protein ◽  
Ras Genes ◽  
Essential Function ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A

1989 ◽  
Vol 9 (6) ◽  
pp. 2453-2463
Author(s):  
P Yaciuk ◽  
J K Choi ◽  
D Shalloway
Keyword(s):  
Protein Kinase ◽  
3T3 Cells ◽  
Triple Mutant ◽  
Dependent Protein Kinase ◽  
Mutant Proteins ◽  
Tetradecanoyl Phorbol Acetate ◽  
Dependent Protein ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.

Both products of the fosB gene, FosB and its short form, FosB/SF, are transcriptional activators in fibroblasts

1991 ◽  
Vol 11 (11) ◽  
pp. 5470-5478
Author(s):  
P Dobrazanski ◽  
T Noguchi ◽  
K Kovary ◽  
C A Rizzo ◽  
P S Lazo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Cycle Progression ◽  
Short Form ◽  
Constitutive Expression ◽  
Inhibitory Effect ◽  
Negative Regulator ◽  
3T3 Cells ◽  
C Terminus ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We demonstrate that a member of the fos family, the fosB gene, gives rise to two transcripts by alternative splicing of exon 4, generating two proteins, FosB of 338 amino acids and a short form, FosB/SF, which contains the DNA binding and dimerization domains but not the 101 amino acids of the C terminus. FosB/SF activates an AP-1-chloramphenicol acetyltransferase construct in NIH 3T3 cells, as determined by transient and stable transfections, although more weakly than does FosB. In contrast to FosB, FosB/SF has lost its ability to repress the dyad symmetry element of the c-fos gene. FosB/SF when expressed in excess to FosB can downmodulate the activity of FosB. Constitutive expression of high levels of FosB/SF in NIH 3T3 cells has no significant inhibitory effect in the induction of cell proliferation or cell cycle progression, indicating that FosB/SF is not a negative regulator of cell growth. This conclusion is further confirmed by the observation that the majority of the Jun molecules are complexed with FosB/SF in the FosB/SF-overexpressing cells.

scholarly journals Leukemia-Related Transcription Factor TEL Is Negatively Regulated through Extracellular Signal-Regulated Kinase-Induced Phosphorylation

2004 ◽  
Vol 24 (8) ◽  
pp. 3227-3237 ◽  
Author(s):  
Kazuhiro Maki ◽  
Honoka Arai ◽  
Kazuo Waga ◽  
Ko Sasaki ◽  
Fumihiko Nakamura ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Effect ◽  
Phosphorylation Sites ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser213 and Ser257. TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser213 and Ser257 functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.

scholarly journals Cell Cycle Progression Regulates Biogenesis and Cellular Localization of Lipid Droplets

2019 ◽  
Vol 39 (9) ◽  
Author(s):  
André L. S. Cruz ◽  
Nina Carrossini ◽  
Leonardo K. Teixeira ◽  
Luis F. Ribeiro-Pinto ◽  
Patricia T. Bozza ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Lipid Accumulation ◽  
Lipid Droplets ◽  
Cell Cycle Progression ◽  
Cellular Transformation ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACTIntracellular lipid accumulation has been associated with a poor prognosis in cancer. We have previously reported the involvement of lipid droplets in cell proliferation in colon cancer cells, suggesting a role for these organelles in cancer development. In this study, we evaluate the role of lipid droplets in cell cycle regulation and cellular transformation. Cell cycle synchronization of NIH 3T3 cells revealed increased numbers and dispersed distribution of lipid droplets specifically during S phase. Also, the transformed cell lineage NIH 3T3-H-rasV12showed an accumulation of both lipid droplets and PLIN2 protein above the levels in NIH 3T3 cells.PLIN2gene overexpression, however, was not able to induce NIH 3T3 cell transformation, disproving the hypothesis thatPLIN2is an oncogene. Furthermore, positive PLIN2 staining was strongly associated with highly proliferative Ki-67-positive areas in human colon adenocarcinoma tissue samples. Taken together, these results indicate that cell cycle progression is associated with tight regulation of lipid droplets, a process that is altered in transformed cells, suggesting the existence of a mechanism that connects cell cycle progression and cell proliferation with lipid accumulation.

scholarly journals Transformation of NIH 3T3 cells by cotransfection with c-src and nuclear oncogenes.

1987 ◽  
Vol 7 (10) ◽  
pp. 3582-3590 ◽  
Author(s):  
D Shalloway ◽  
P J Johnson ◽  
E O Freed ◽  
D Coulter ◽  
W A Flood
Keyword(s):  
3T3 Cells ◽  
Focus Formation ◽  
Adenovirus E1a ◽  
Rous Sarcoma ◽  
Cellular Homolog ◽  
Sarcoma Virus ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

pp60c-src, the cellular homolog of the Rous sarcoma virus transforming protein, does not completely transform cells even when present at high levels, but has been shown to be involved in polyomavirus-induced transformation when activated by polyomavirus middle T (pmt)-antigen binding. Here we show that cotransfection, but not solo transfection, of expression plasmids for c-src and either adenovirus E1A, v-myc, c-myc, or the 5' half of polyomavirus large T (pltN) antigen into NIH 3T3 cells induces anchorage-independent growth, enhanced focus formation, and, for pltN cotransfection, tumorigenicity in adult NFS mice. Enhancement of transformation was not observed with polyomavirus small t (pst) antigen. Cotransfection of c-src with pltN induced modification of pp60c-src that altered its electrophoretic mobility and in vivo phosphorylation state and stimulated its in vitro kinase activity. Similar alterations were not seen after c-src-E1A cotransfection, suggesting that at least two different mechanisms of enhancement are involved.

scholarly journals Regulation of Calreticulin Gene Expression by Calcium

1997 ◽  
Vol 138 (3) ◽  
pp. 547-557 ◽  
Author(s):  
Mathilde Waser ◽  
Nasrin Mesaeli ◽  
Charlotte Spencer ◽  
Marek Michalak
Keyword(s):  
Gene Expression ◽  
Genomic Dna ◽  
Chloramphenicol Acetyltransferase ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Transcription Analysis ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We have isolated and characterized a 12-kb mouse genomic DNA fragment containing the entire calreticulin gene and 2.14 kb of the promoter region. The mouse calreticulin gene consists of nine exons and eight introns, and it spans 4.2 kb of genomic DNA. A 1.8-kb fragment of the calreticulin promoter was subcloned into a reporter gene plasmid containing chloramphenicol acetyltransferase. This construct was then used in transient and stable transfection of NIH/ 3T3 cells. Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. Transactivation of the calreticulin promoter was also increased by fourfold in NIH/3T3 cells treated with bradykinin, a hormone that induces Ca2+ release from the intracellular Ca2+ stores. Analysis of the promoter deletion constructs revealed that A23187- and thapsigargin-responsive regions are confined to two regions (−115 to −260 and −685 to −1,763) in the calreticulin promoter that contain the CCAAT nucleotide sequences. Northern blot analysis of cells treated with A23187, or with thapsigargin, revealed a fivefold increase in calreticulin mRNA levels. Thapsigargin also induced a fourfold increase in calreticulun protein levels. Importantly, we show by nuclear run-on transcription analysis that calreticulin gene transcription is increased in NIH/3T3 cells treated with A23187 and thapsigargin in vivo. This increase in gene expression required over 4 h of continuous incubation with the drugs and was also sensitive to treatment with cycloheximide, suggesting that it is dependent on protein synthesis. Changes in the concentration of extracellular and cytoplasmic Ca2+ did not affect the increased expression of the calreticulin gene. These studies suggest that stress response to the depletion of intracellular Ca2+ stores induces expression of the calreticulin gene in vitro and in vivo.

scholarly journals Induction of Syncytia by Neuropathogenic Murine Leukemia Viruses Depends on Receptor Density, Host Cell Determinants, and the Intrinsic Fusion Potential of Envelope Protein

1999 ◽  
Vol 73 (11) ◽  
pp. 9377-9385 ◽  
Author(s):  
Maeran Chung ◽  
Krishnakumar Kizhatil ◽  
Lorraine M. Albritton ◽  
Glen N. Gaulton
Keyword(s):  
Host Cell ◽  
Cell Fusion ◽  
Syncytium Formation ◽  
Receptor Expression ◽  
Receptor Density ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Murine Leukemia ◽  
Nih 3T3 Cells

ABSTRACT Infection by the neuropathogenic murine leukemia virus (MLV) TR1.3 results in hemorrhagic disease that correlates directly to in vivo syncytium formation of brain capillary endothelial cells (BCEC). This phenotype maps to amino acid 102 in the envelope (Env) protein of TR1.3. Substitution of glycine (G) for tryptophan (W) at this position (W102G Env) in the nonpathogenic MLV FB29 induces both syncytium formation and neurologic disease in vivo. Using an in vitro gene reporter cell fusion assay, we showed that fusion either with murine NIH 3T3 cells or with nonmurine target cells that expressed receptors at or below endogenous murine levels mirrored that seen in BCEC in vivo. In these instances only TR1.3 and W102G Env induced cell fusion. In contrast, when receptor levels on nonmurine cells were raised above endogenous murine levels, FB29 Env was as fusogenic as the neuropathogenic TR1.3 and W102G Env. These results indicate that TR1.3 Env and W102G Env are intrinsically more fusogenic than FB29 Env, that the induction of fusion requires a threshold number of receptors that is greater for FB29 Env than for TR1.3 or W102G Env, and that receptor density on murine NIH 3T3 cells and BCEC is below the threshold for FB29-dependent fusion. Surprisingly, receptor density on NIH 3T3 cells could not be increased by stable expression of exogenous receptors, and FB29-dependent fusion was not observed in NIH 3T3 cells that transiently expressed elevated receptor numbers. These results suggest that an additional undefined host cell factor(s) may limit both receptor expression and fusion potential in murine cells.

Sign in / Sign up

Export Citation Format

Share Document