The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein

1991 ◽  
Vol 11 (3) ◽  
pp. 1523-1530
Author(s):  
J E Buss ◽  
L A Quilliam ◽  
K Kato ◽  
P J Casey ◽  
P A Solski ◽  
...  
Keyword(s):  
Biological Activities ◽  
Chimeric Protein ◽  
Cell System ◽  
Full Length ◽  
Ras Proteins ◽  
3T3 Cells ◽  
Farnesyl Transferase ◽  
Oncogenic Ras ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

scholarly journals The COOH-terminal domain of the Rap1A (Krev-1) protein is isoprenylated and supports transformation by an H-Ras:Rap1A chimeric protein.

1991 ◽  
Vol 11 (3) ◽  
pp. 1523-1530 ◽  
Author(s):  
J E Buss ◽  
L A Quilliam ◽  
K Kato ◽  
P J Casey ◽  
P A Solski ◽  
...  
Keyword(s):  
Biological Activities ◽  
Chimeric Protein ◽  
Cell System ◽  
Full Length ◽  
Ras Proteins ◽  
3T3 Cells ◽  
Farnesyl Transferase ◽  
Oncogenic Ras ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Although the Rap1A protein resembles the oncogenic Ras proteins both structurally and biochemically, Rap1A exhibits no oncogenic properties. Rather, overexpression of Rap1A can reverse Ras-induced transformation of NIH 3T3 cells. Because the greatest divergence in amino acid sequence between Ras and Rap1A occurs at the COOH terminus, the role of this domain in the opposing biological activities of these proteins was examined. COOH-terminal processing and membrane association of Rap1A were studied by constructing and expressing a chimeric protein (composed of residues 1 to 110 of an H-Ras activated by a Leu-61 mutation attached to residues 111 to 184 of Rap1A) in NIH 3T3 cells and a full-length human Rap1A protein in a baculovirus-Sf9 insect cell system. Both the chimeric protein and the full-length protein were synthesized as a 23-kDa cytosolic precursor that rapidly bound to membranes and was converted into a 22-kDa form that incorporated label derived from [3H]mevalonate. The mature 22-kDa form also contained a COOH-terminal methyl group. Full-length Rap1A, expressed in insect cells, was modified by a C20 (geranylgeranyl) isoprenoid. In contrast, H-Ras, expressed in either Sf9 insect or NIH 3T3 mouse cells contained a C15 (farnesyl) group. This suggests that the Rap1A COOH terminus is modified by a prenyl transferase that is distinct from the farnesyl transferase that modifies Ras proteins. Nevertheless, in NIH 3T3 cells the chimeric Ras:Rap1A protein retained the transforming activity conferred by the NH2-terminal Ras61L domain. This demonstrates that the modifications and localization signals of the COOH terminus of Rap1A can support the interactions between H-Ras and membranes that are required for transformation.

A full-length murine 2-5A synthetase cDNA transfected in NIH-3T3 cells impairs EMCV but not VSV replication

Virology ◽  
1990 ◽  
Vol 179 (1) ◽  
pp. 228-233 ◽  
Author(s):  
Eliana M. Coccia ◽  
Giovanna Romeo ◽  
Ahuva Nissim ◽  
Giovanna Marziali ◽  
Roberto Albertini ◽  
...  
Keyword(s):  
Full Length ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

1992 ◽  
Vol 12 (6) ◽  
pp. 2606-2615 ◽  
Author(s):  
A D Cox ◽  
M M Hisaka ◽  
J E Buss ◽  
C J Der
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Biological Activities ◽  
Ras Proteins ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Similar Growth ◽  
Growth Inhibiting

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.

Oncogenic Transformation of NIH 3T3 Fibroblasts by BCR-ABL Oncoprotein Requires the IL-3 Receptor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3370-3370
Author(s):  
Wenjing Tao ◽  
Hui Lin ◽  
Tong Sun ◽  
Ajoy K. Samanta ◽  
Ralph B. Arlinghaus
Keyword(s):  
Kinase Inhibitor ◽  
Philadelphia Chromosome ◽  
Cell System ◽  
Receptor Expression ◽  
Oncogenic Transformation ◽  
3T3 Cells ◽  
Lymphoid Leukemia ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Abstract Bcr-Abl is a leukemia-inducing protein, which causes oncogenic transformation of myeloid progenitors in Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and lymphoid progenitors in Ph+ acute lymphoid leukemia (ALL). Oncogenic transformation of hematopoietic cells by the Bcr-Abl oncoprotein directly involves the activation Jak2 tyrosine kinase and the Stat5 transcription factor. Both proteins are normally linked to the IL-3/GM-CSF receptors for growth and survival. Since fibroblastic cells are not targets of BCR-ABL induced oncogenesis, we determined whether forced expression of the IL-3 receptor would allow oncogenic transformation of NIH 3T3 fibroblasts known to be resistant to transformation by BCR-ABL. NIH 3T3 cells transduced with the human IL-3 receptor a and b chains were highly susceptible to oncogenic transformation by expression of BCR-ABL. Forced expression of both receptor chains but not either one alone allowed efficient foci formation of NIH 3T3 cells expressing BCR-ABL, and these cells formed colonies in soft agar whereas BCR-ABL positive NIH 3T3 cells lacking IL-3 receptor expression did not. The Bcr-Abl kinase inhibitor imatinib mesylate (1 mM) and the Jak kinase inhibitor AG490 (10 mM) strongly inhibited agar colony formation. A small molecule inhibitor of Jak2 kinase, 1,2,3,4,5,6-hexabromocyclohexane reported to be specific for Jak2 (Sandberg et al. J. Med. Chem, 2005)-significantly reduced the phosphorylation of Gab2 at the YxxM motif, which is needed for activation of the PI-3 kinase and Akt, two proteins that are part of the Bcr-Abl/Jak2 Network (Samanta et al. Cancer Res, 2006). These findings indicate that Bcr-Abl oncoprotein requires the IL-3 receptor/Jak2/Stat5 pathways for oncogenic transformation of NIH 3T3 fibroblasts, and may explain partially why Bcr-Abl oncogenesis is restricted to hematopoietic malignancies. Furthermore, this cell system in fibroblastic and other cell lineages will provide a model to probe the detailed steps that require IL-3 receptor and Jak2 for Bcr-Abl induced leukemia.

scholarly journals Overexpression of core-binding factor alpha (CBF alpha) reverses cellular transformation by the CBF beta-smooth muscle myosin heavy chain chimeric oncoprotein.

1995 ◽  
Vol 15 (9) ◽  
pp. 4980-4989 ◽  
Author(s):  
A Hajra ◽  
P P Liu ◽  
N A Speck ◽  
F S Collins
Keyword(s):  
Smooth Muscle ◽  
Chimeric Protein ◽  
Cellular Transformation ◽  
3T3 Cells ◽  
Dna Complexes ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Nih 3T3 ◽  
Muscle Myosin ◽  
Nih 3T3 Cells

A fusion between the transcription factor core-binding factor beta (CBF beta; also known as PEBP2 beta) and the tail region of smooth muscle myosin heavy chain (SMMHC) is generated by an inversion of chromosome 16 [inv(16) (p13q22)] associated with the M4Eo subtype of acute myeloid leukemia. We have previously shown that this CBF beta-SMMHC chimeric protein can transform NIH 3T3 cells and that this process requires regions of the chimeric protein necessary for association with the CBF alpha subunit. In this study, we show that NIH 3T3 cells overexpressing murine Cbf alpha 2 (also known as Aml1) cannot be transformed by CBF beta-SMMHC and that overexpression of Cbf alpha 2 in cells previously transformed by CBF beta-SMMHC reverts the cells to a less transformed phenotype. Cbf alpha 2 overexpression does not cause any gross morphological changes to NIH 3T3 cells but does result in increased CBF activity, as indicated by electrophoretic mobility shift assays and transactivation of reporter constructs. Cells transformed by CBF beta-SMMHC lack normal CBF-DNA complexes and have decreased levels of transactivation. Reversion of CBF beta-SMMHC transformation by Cbf alpha 2 is associated with a restoration of normal CBF-DNA complexes and transactivation activity. A Cbf alpha 2 mutant lacking transactivation properties does not transform cells when overexpressed, nor does it protect cells from CBF beta-SMMHC transformation. These results suggest that CBF beta-SMMHC interferes with the normal function of CBF and that this interference is necessary but not sufficient for cellular transformation.

scholarly journals The Saccharomyces cerevisiae SDC25 C-domain gene product overcomes the dominant inhibitory activity of Ha-Ras Asn-17.

1993 ◽  
Vol 13 (1) ◽  
pp. 39-43 ◽  
Author(s):  
F Schweighoffer ◽  
H Cai ◽  
M C Chevallier-Multon ◽  
I Fath ◽  
G Cooper ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Ras Proteins ◽  
3T3 Cells ◽  
Kinase Gene ◽  
Inhibition Of Proliferation ◽  
Negative Effect ◽  
Carboxy Terminal ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The carboxy-terminal part of the Saccharomyces cerevisiae SDC25 gene product (SDC25 C domain) can elicit activation of mammalian Ras proteins. Specifically, SDC25 C domain functions as an exchange factor for cellular Ras proteins in CHO cells. In this study, we used the dominant inhibitory Ha-Ras Asn-17 mutant and SDC25 C domain to further investigate the interaction between cellular Ras proteins and their putative endogenous guanine nucleotide-releasing factors. Transcription from the polyomavirus thymidine kinase gene (Py tk) promoter is strongly inhibited by the expression of Ha-Ras Asn-17 in NIH 3T3 cells. Coexpression of SDC25 C domain overcomes the negative effect of the Ras mutant on the Py tk promoter. On the other hand, transactivation of the Ras-responsive element of the Py tk promoter induced by SDC25 C domain is lost upon coexpression of increasing amounts of Ha-Ras Asn-17. In addition, coexpression of SDC25 C domain overcomes the inhibition of proliferation of NIH 3T3 cells caused by Ha-Ras Asn-17. These results are consistent with the idea that the Ha-Ras Asn-17 mutant functions by titrating an upstream activator of cellular Ras proteins.

scholarly journals Compartmentalized Ras Proteins Transform NIH 3T3 Cells with Different Efficiencies

2010 ◽  
Vol 31 (5) ◽  
pp. 983-997 ◽  
Author(s):  
C.-M. Cheng ◽  
H. Li ◽  
S. Gasman ◽  
J. Huang ◽  
R. Schiff ◽  
...  
Keyword(s):  
Ras Proteins ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Mechanisms of oncogene-mediated alterations in metastatic ability

1992 ◽  
Vol 70 (9) ◽  
pp. 817-821 ◽  
Author(s):  
Ann F. Chambers
Keyword(s):  
Signal Transduction ◽  
Calcium Binding ◽  
Proteolytic Enzymes ◽  
Model Systems ◽  
3T3 Cells ◽  
Altered Expression ◽  
Oncogenic Ras ◽  
Expression Of Genes ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Transfected ras oncogenes have been shown to induce metastatic properties in some cells. This altered behavior is likely due to changes in ras-mediated signal transduction pathways, resulting in altered expression of genes important to metastasis. Clarification of the mechanisms by which ras is able to induce metastatic ability in model systems will improve our understanding of tumor progression, even in those cells in which ras activation has not been implicated. Many of the consequences of ras expression also have been detected in cells that have become metastatic in the absence of altered ras, suggesting that there is a set of common changes that can lead to metastasis, with multiple signals capable of eliciting these changes. We have identified several changes in metastatic, ras-transformed NIH 3T3 cells that may contribute to their increased malignancy, including expression of proteolytic enzymes and their inhibitors, and adhesive and calcium-binding proteins. Not all cells, however, respond in this way to expression of oncogenic ras. We have found that murine LTA cells, which are tumorigenic but nonmetastatic, are ras resistant and remain nonmetastatic when expressing high levels of transfected ras, in contrast to NIH 3T3 cells, which are ras sensitive and become both tumorigenic and metastatic in response to comparable levels of ras. LTA cells differ in their patterns of gene expression in response to ras when compared with NIH 3T3 cells, suggesting that the two cell lines process the ras signal differently. Here we review our results with ras-transfected NIH 3T3 and LTA cells. An understanding of the ways in which these cells differ in their response to an oncogenic signal may provide clues to mechanisms of tumor progression.Key words: ras, signal transduction, proteolytic enzymes, osteopontin, calcyclin.

scholarly journals Self-Assembled Thermoresponsive Nanogel from Grafted Hyaluronic Acid as a Biocompatible Delivery Platform for Curcumin with Enhanced Drug Loading and Biological Activities

Polymers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 194
Author(s):  
Jittima Amie Luckanagul ◽  
Pahweenvaj Ratnatilaka Na Bhuket ◽  
Chawanphat Muangnoi ◽  
Pranee Rojsitthisak ◽  
Qian Wang ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Biological Activities ◽  
Drug Loading ◽  
Aqueous Solubility ◽  
3T3 Cells ◽  
Delivery Platform ◽  
Model Drug ◽  
Equivalence Model ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

A hyaluronic acid-grafted poly(N-isopropylacrylamide) (HA-pNIPAM) was synthesized as a polymeric nanogel platform for encapsulation and delivery of hydrophobic bioactive compounds using curcumin as a model drug. As demonstrated by transmission electron microscopy and dynamic light scattering techniques, the HA-pNIPAM was simply assembled into spherical nano-sized particles with the thermoresponsive behavior. The success of curcumin aqueous solubilization was confirmed by fluorescent spectroscopy. The resulting nanogel formulation enhanced the aqueous solubility and uptake into NIH-3T3 cells of curcumin. This nanogel formulation also demonstrates cytocompatibility against NIH-3T3 cells, which deems it safe as a delivery vehicle. Moreover, the formulation has a slight skin-protection effect using an artificial skin equivalence model. The curcumin-loaded HA-pNIPAM nanogel showed an anti-proliferative activity against MDA-MB-231, Caco-2, HepG2, HT-29, and TNF-α-induced hyperproliferation of keratinocyte (HaCaT) cells. The thermoresponsive HA-pNIPAM nanogel reported here could be further optimized as a platform for controlled-release systems to encapsulate pharmaceuticals for therapeutic applications.

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein

1992 ◽  
Vol 12 (6) ◽  
pp. 2606-2615
Author(s):  
A D Cox ◽  
M M Hisaka ◽  
J E Buss ◽  
C J Der
Keyword(s):  
Mammalian Cells ◽  
Protein Modification ◽  
Biological Activities ◽  
Ras Proteins ◽  
Ras Protein ◽  
Oncogenic Ras ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Similar Growth ◽  
Growth Inhibiting

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.

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