Regulation of a Heterologous Glucose Transporter Promoter in Chicken Embryo Fibroblasts

1998 ◽  
Vol 252 (2) ◽  
pp. 318-323 ◽  
Author(s):  
Sarah E. Steane ◽  
Dawn Mylott ◽  
Martyn K. White
Keyword(s):  
Chicken Embryo ◽  
Glucose Transporter ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Differential regulation of glucose transporter isoforms by the src oncogene in chicken embryo fibroblasts

1991 ◽  
Vol 11 (9) ◽  
pp. 4448-4454
Author(s):  
M K White ◽  
T B Rall ◽  
M J Weber
Keyword(s):  
Glucose Transport ◽  
Chicken Embryo ◽  
Glucose Transporter ◽  
Sequence Similarity ◽  
Transporter Protein ◽  
Mrna Induction ◽  
Embryo Fibroblasts ◽  
Type 3 ◽  
Chicken Embryo Fibroblasts

The increase in glucose transport that occurs when chicken embryo fibroblasts (CEFs) are transformed by src is associated with an increase in the amount of type 1 glucose transporter protein, and we have previously shown that this effect is due to a decrease in the degradation rate of this protein. The rate of CEF type 1 glucose transporter biosynthesis and the level of its mRNA are unaffected by src transformation. To study the molecular basis of this phenomenon, we have been isolating chicken glucose transporter cDNAs by hybridization to a rat type 1 glucose transporter probe at low stringency. Surprisingly, these clones corresponded to a message encoding a protein which has most sequence similarity to the human type 3 glucose transporter and which we refer to as CEF-GT3. CEF-GT3 is clearly distinct from the CEF type 1 transporter that we have previously described. Northern (RNA) analysis of CEF RNA with CEF-GT3 cDNA revealed two messages of 1.7 and 3.3 kb which were both greatly induced by src transformation. When the CEF-GT3 cDNA was expressed in rat fibroblasts, a three-to fourfold enhancement of 2-deoxyglucose uptake was observed, indicating that CEF-GT3 is a functional glucose transporter. Northern analyses using a CEF-GT3 and a rat type 1 probe demonstrated that there is no hybridization between different isoforms but that there is cross-species hybridization between the rat type 1 probe and the chicken homolog. Southern blot analyses confirmed that the chicken genomic type 1 and type 3 transporters are encoded by distinct genes. We conclude that CEFs express two types of transporter, type 1 (which we have previously reported to be regulated posttranslationally by src) and a novel type 3 isoform which, unlike type 1, shows mRNA induction upon src transformation. We conclude that src regulates glucose transport in CEFs simultaneously by two different mechanisms.

Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms

1988 ◽  
Vol 8 (1) ◽  
pp. 138-144
Author(s):  
M K White ◽  
M J Weber
Keyword(s):  
Cell Line ◽  
Glucose Transport ◽  
Chicken Embryo ◽  
Glucose Transporter ◽  
Mrna Levels ◽  
Primary Fibroblast ◽  
Transporter Protein ◽  
Embryo Fibroblasts ◽  
The Difference ◽  
Chicken Embryo Fibroblasts

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.

scholarly journals Characterization of the avian GLUT1 glucose transporter: differential regulation of GLUT1 and GLUT3 in chicken embryo fibroblasts.

1995 ◽  
Vol 6 (11) ◽  
pp. 1575-1589 ◽  
Author(s):  
P Wagstaff ◽  
H Y Kang ◽  
D Mylott ◽  
P J Robbins ◽  
M K White
Keyword(s):  
Glucose Transport ◽  
Chicken Embryo ◽  
Mammalian Cells ◽  
Glucose Transporter ◽  
Sequence Similarity ◽  
Tumor Promoter ◽  
Amino Acid Sequence Similarity ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Glut1 Glucose Transporter

Vertebrate cells that are transformed by oncogenes such as v-src or are stimulated by mitogens have increased rates of glucose uptake. In rodent cells, the mechanisms whereby glucose transport is up-regulated are well understood. Stimulation of glucose transport involves an elevation in mRNA encoding the GLUT1 glucose transporter that is controlled at the levels of both transcription and mRNA stability. Cloning and sequencing of chicken GLUT1 cDNA showed that it shares 95% amino acid sequence similarity to mammalian GLUT1s. Nevertheless, unlike mammalian GLUT1 mRNA, it was not induced by v-src, serum addition, or treatment with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in chicken embryo fibroblasts. Rather, the induction of glucose transport in chicken embryo fibroblasts by v-src, serum, and 12-O-tetradecanoylphorbol 13-acetate was associated with induction of GLUT3 mRNA level and GLUT3 transcription. Rat fibroblasts were also found to express both GLUT1 and GLUT3 isoforms, but v-src induced GLUT1 and not GLUT3. This suggests that animal cells require both a basal and an upregulatable glucose transporter and that these functions have been subsumed by different GLUT isoforms in avian and mammalian cells.

scholarly journals Differential regulation of glucose transporter isoforms by the src oncogene in chicken embryo fibroblasts.

1991 ◽  
Vol 11 (9) ◽  
pp. 4448-4454 ◽  
Author(s):  
M K White ◽  
T B Rall ◽  
M J Weber
Keyword(s):  
Glucose Transport ◽  
Chicken Embryo ◽  
Glucose Transporter ◽  
Sequence Similarity ◽  
Transporter Protein ◽  
Mrna Induction ◽  
Embryo Fibroblasts ◽  
Type 3 ◽  
Chicken Embryo Fibroblasts

The increase in glucose transport that occurs when chicken embryo fibroblasts (CEFs) are transformed by src is associated with an increase in the amount of type 1 glucose transporter protein, and we have previously shown that this effect is due to a decrease in the degradation rate of this protein. The rate of CEF type 1 glucose transporter biosynthesis and the level of its mRNA are unaffected by src transformation. To study the molecular basis of this phenomenon, we have been isolating chicken glucose transporter cDNAs by hybridization to a rat type 1 glucose transporter probe at low stringency. Surprisingly, these clones corresponded to a message encoding a protein which has most sequence similarity to the human type 3 glucose transporter and which we refer to as CEF-GT3. CEF-GT3 is clearly distinct from the CEF type 1 transporter that we have previously described. Northern (RNA) analysis of CEF RNA with CEF-GT3 cDNA revealed two messages of 1.7 and 3.3 kb which were both greatly induced by src transformation. When the CEF-GT3 cDNA was expressed in rat fibroblasts, a three-to fourfold enhancement of 2-deoxyglucose uptake was observed, indicating that CEF-GT3 is a functional glucose transporter. Northern analyses using a CEF-GT3 and a rat type 1 probe demonstrated that there is no hybridization between different isoforms but that there is cross-species hybridization between the rat type 1 probe and the chicken homolog. Southern blot analyses confirmed that the chicken genomic type 1 and type 3 transporters are encoded by distinct genes. We conclude that CEFs express two types of transporter, type 1 (which we have previously reported to be regulated posttranslationally by src) and a novel type 3 isoform which, unlike type 1, shows mRNA induction upon src transformation. We conclude that src regulates glucose transport in CEFs simultaneously by two different mechanisms.

scholarly journals Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms.

1988 ◽  
Vol 8 (1) ◽  
pp. 138-144 ◽  
Author(s):  
M K White ◽  
M J Weber
Keyword(s):  
Cell Line ◽  
Glucose Transport ◽  
Chicken Embryo ◽  
Glucose Transporter ◽  
Mrna Levels ◽  
Primary Fibroblast ◽  
Transporter Protein ◽  
Embryo Fibroblasts ◽  
The Difference ◽  
Chicken Embryo Fibroblasts

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.

The src oncogene can regulate a human glucose transporter expressed in chicken embryo fibroblasts

1990 ◽  
Vol 10 (4) ◽  
pp. 1301-1306
Author(s):  
M K White ◽  
M J Weber
Keyword(s):  
Chicken Embryo ◽  
Glucose Transporter ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Transporter Protein ◽  
Chicken Cell ◽  
Two Temperatures ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

When fibroblasts are transformed by the src oncogene, there is a two- to fivefold increase in glucose transport and in the level of immunoprecipitable glucose transporter protein. In chicken embryo fibroblasts (CEFs), this increase is correlated with a comparable reduction in the rate at which the glucose transporter protein is turned over. In contrast, in mammalian fibroblasts glucose transporter biosynthesis is increased by src, but there is little or no change in its turnover. To further understand the action of src on transporter turnover, we investigated whether a mammalian transporter can be stabilized by src in a chicken cell environment. The human type 1 glucose transporter protein (hGT), originally cloned from HepG2 cells, was expressed in CEFs or Rat-1 fibroblasts by using a retroviral vector. In CEFs transformed by a temperature-sensitive src mutant, tsNY68, turnover of hGT was lower at the permissive temperature (36 degrees C) than at the nonpermissive temperature (42 degrees C). When this protein was expressed in CEFs transformed by wild-type src, no difference in turnover was observed at the two temperatures. In the case of Rat-1 cells transformed by the temperature-sensitive src mutant tsLA29, turnover of hGT was the same at the permissive temperature (35 degrees C) as at the nonpermissive temperature (39.5 degrees C). These data demonstrate that a heterologous glucose transporter behaves in the same way in chicken and rat cells as the respective endogenous transporter, i.e., when src is active, the protein is stablilized against turnover in chicken cells but not in rat cells.

scholarly journals The src oncogene can regulate a human glucose transporter expressed in chicken embryo fibroblasts.

1990 ◽  
Vol 10 (4) ◽  
pp. 1301-1306 ◽  
Author(s):  
M K White ◽  
M J Weber
Keyword(s):  
Chicken Embryo ◽  
Glucose Transporter ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Transporter Protein ◽  
Chicken Cell ◽  
Two Temperatures ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

When fibroblasts are transformed by the src oncogene, there is a two- to fivefold increase in glucose transport and in the level of immunoprecipitable glucose transporter protein. In chicken embryo fibroblasts (CEFs), this increase is correlated with a comparable reduction in the rate at which the glucose transporter protein is turned over. In contrast, in mammalian fibroblasts glucose transporter biosynthesis is increased by src, but there is little or no change in its turnover. To further understand the action of src on transporter turnover, we investigated whether a mammalian transporter can be stabilized by src in a chicken cell environment. The human type 1 glucose transporter protein (hGT), originally cloned from HepG2 cells, was expressed in CEFs or Rat-1 fibroblasts by using a retroviral vector. In CEFs transformed by a temperature-sensitive src mutant, tsNY68, turnover of hGT was lower at the permissive temperature (36 degrees C) than at the nonpermissive temperature (42 degrees C). When this protein was expressed in CEFs transformed by wild-type src, no difference in turnover was observed at the two temperatures. In the case of Rat-1 cells transformed by the temperature-sensitive src mutant tsLA29, turnover of hGT was the same at the permissive temperature (35 degrees C) as at the nonpermissive temperature (39.5 degrees C). These data demonstrate that a heterologous glucose transporter behaves in the same way in chicken and rat cells as the respective endogenous transporter, i.e., when src is active, the protein is stablilized against turnover in chicken cells but not in rat cells.

scholarly journals Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

1985 ◽  
Vol 100 (3) ◽  
pp. 692-703 ◽  
Author(s):  
J J Lin ◽  
D M Helfman ◽  
S H Hughes ◽  
C S Chou
Keyword(s):  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Actin Binding ◽  
Transformed Cells ◽  
Two Dimensional ◽  
Tropomyosin Isoforms ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed cells.

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