Plasminogen activator: analysis of enzyme induction by ultraviolet irradiation mapping

1981 ◽  
Vol 1 (10) ◽  
pp. 884-890
Author(s):  
R Miskin ◽  
E Reich ◽  
K Dixon
Keyword(s):  
Plasminogen Activator ◽  
Cross Sections ◽  
Ultraviolet Irradiation ◽  
Chicken Embryo ◽  
Phorbol Esters ◽  
Transformed Cells ◽  
Normal Cells ◽  
Embryo Fibroblasts ◽  
Mapping Techniques ◽  
Chicken Embryo Fibroblasts

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agents induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.

scholarly journals Plasminogen activator: analysis of enzyme induction by ultraviolet irradiation mapping.

1981 ◽  
Vol 1 (10) ◽  
pp. 884-890
Author(s):  
R Miskin ◽  
E Reich ◽  
K Dixon
Keyword(s):  
Plasminogen Activator ◽  
Cross Sections ◽  
Ultraviolet Irradiation ◽  
Chicken Embryo ◽  
Phorbol Esters ◽  
Transformed Cells ◽  
Normal Cells ◽  
Embryo Fibroblasts ◽  
Mapping Techniques ◽  
Chicken Embryo Fibroblasts

Ultraviolet irradiation mapping techniques have previously been used to study the organization of eucaryotic gene classes and transcription units. We used the same method to probe some regulatory phenomena observed in the induction of plasminogen activator (PA) biosynthesis: PA synthesis in chicken embryo fibroblasts is induced by tumor-promoting phorbol esters and by retinoic acid; furthermore, PA induction by phorbol esters is synergistic with transformation, being 10- to 20-fold greater in virus-transformed cells than in normal cells. We found that the ultraviolet irradiation inactivation cross sections for PA induction by phorbol esters and by retinoate differed significantly, suggesting that these agents induce PA biosynthesis by different mechanisms. On the other hand, the ultraviolet irradiation sensitivity of phorbol ester induction in normal chicken embryo fibroblasts was the same as in transformed cells, indicating that the synergism of transformation and phorbol esters is probably not due to different pathways of PA induction.

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.

scholarly journals Identification of Phosphotyrosine-Containing Proteins in Untransformed and Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts

1982 ◽  
Vol 2 (6) ◽  
pp. 653-665 ◽  
Author(s):  
Ricardo Martinez ◽  
Kenji D. Nakamura ◽  
Michael J. Weber
Keyword(s):  
Protein Kinases ◽  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Cellular Protein ◽  
Transformed Cells ◽  
Phosphoamino Acid ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Phosphorylation on tyrosine residues mediated by pp60srcappears to be a primary biochemical event leading to the establishment of the transformed phenotype in Rous sarcoma virus (RSV)-infected cells. To identify the cellular proteins that undergo tyrosine phosphorylation during transformation, a32P-labeled RSV-transformed chicken embryo cell extract was analyzed by electrophoresis on a polyacrylamide gel. After slicing the gel into approximately 60 slices, phosphoamino acid analyses were carried out on the protein recovered from each gel slice. Phosphotyrosine was found in every gel slice, with two major peaks of this phosphoamino acid aroundMr's of 59 and 36 kilodaltons. When the same analysis was performed with cells infected with a transformation-defectivesrcdeletion mutant of RSV (tdNY101), significant and reproducible peaks of phosphotyrosine were found in only 2 of 60 gel slices. These gel slices corresponded toMr's of 42 and 40 kilodaltons. Identical results were obtained with normal uninfected chicken embryo fibroblasts. We conclude from these observations that pp60srcor the combined action of pp60srcand pp60src-activated cellular protein kinases cause the tyrosine-specific phosphorylation of a very large number of cellular polypeptides in RSV-transformed cells. In addition, untransformed cells appear to possess one or more active tyrosine-specific protein kinases which are responsible for the phosphorylation of a limited number of proteins. These proteins are different from the major phosphotyrosine-containing proteins of the transformed cells.

In vivo effect of sodium orthovanadate on pp60c-src kinase

1987 ◽  
Vol 7 (3) ◽  
pp. 1139-1147
Author(s):  
J W Ryder ◽  
J A Gordon
Keyword(s):  
Tyrosine Kinase ◽  
Chicken Embryo ◽  
Sodium Orthovanadate ◽  
Normal Cells ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.

scholarly journals Biosynthesis of calmodulin in normal and virus-transformed chicken embryo fibroblasts.

1984 ◽  
Vol 4 (5) ◽  
pp. 883-889 ◽  
Author(s):  
J G Zendegui ◽  
R E Zielinski ◽  
D M Watterson ◽  
L J Van Eldik
Keyword(s):  
Chicken Embryo ◽  
Chicken Embryo Fibroblast ◽  
Cdna Probe ◽  
Transformed Cells ◽  
Synthesis Rate ◽  
Oncogenic Viruses ◽  
Transformed Fibroblasts ◽  
Significant Difference ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

We report here that the higher levels of calmodulin in transformed chicken embryo fibroblasts are due to an increase in the rate of synthesis of calmodulin that results from an increased amount of calmodulin-specific mRNA in transformed cells. Transformation of several types of eucaryotic cells by oncogenic viruses results in a two- to threefold increase in the intracellular levels of calmodulin. We used the normal chicken embryo fibroblast and its Rous sarcoma virus-transformed counterpart to examine the biosynthesis of calmodulin. We show that the higher levels of calmodulin found in transformed fibroblasts appear to be the consequence of a selective increase in the rate of synthesis of calmodulin above that of total soluble or total cellular protein. A significant difference in the rate of degradation of calmodulin or total protein between transformed and normal cells was not detected. We also examined the mechanism of the increased synthesis rate of calmodulin and show that the levels of calmodulin mRNA are increased in transformed fibroblasts as measured by both translational activity and hybridization to a calmodulin cDNA probe. It is suggested by these data that the higher levels of calmodulin in transformed cells may result from a specific increase in the rate of either calmodulin gene transcription or mRNA processing.

Biosynthesis of calmodulin in normal and virus-transformed chicken embryo fibroblasts

1984 ◽  
Vol 4 (5) ◽  
pp. 883-889
Author(s):  
J G Zendegui ◽  
R E Zielinski ◽  
D M Watterson ◽  
L J Van Eldik
Keyword(s):  
Chicken Embryo ◽  
Chicken Embryo Fibroblast ◽  
Cdna Probe ◽  
Transformed Cells ◽  
Synthesis Rate ◽  
Oncogenic Viruses ◽  
Transformed Fibroblasts ◽  
Significant Difference ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

We report here that the higher levels of calmodulin in transformed chicken embryo fibroblasts are due to an increase in the rate of synthesis of calmodulin that results from an increased amount of calmodulin-specific mRNA in transformed cells. Transformation of several types of eucaryotic cells by oncogenic viruses results in a two- to threefold increase in the intracellular levels of calmodulin. We used the normal chicken embryo fibroblast and its Rous sarcoma virus-transformed counterpart to examine the biosynthesis of calmodulin. We show that the higher levels of calmodulin found in transformed fibroblasts appear to be the consequence of a selective increase in the rate of synthesis of calmodulin above that of total soluble or total cellular protein. A significant difference in the rate of degradation of calmodulin or total protein between transformed and normal cells was not detected. We also examined the mechanism of the increased synthesis rate of calmodulin and show that the levels of calmodulin mRNA are increased in transformed fibroblasts as measured by both translational activity and hybridization to a calmodulin cDNA probe. It is suggested by these data that the higher levels of calmodulin in transformed cells may result from a specific increase in the rate of either calmodulin gene transcription or mRNA processing.

scholarly journals In vivo effect of sodium orthovanadate on pp60c-src kinase.

1987 ◽  
Vol 7 (3) ◽  
pp. 1139-1147 ◽  
Author(s):  
J W Ryder ◽  
J A Gordon
Keyword(s):  
Tyrosine Kinase ◽  
Chicken Embryo ◽  
Sodium Orthovanadate ◽  
Normal Cells ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.

scholarly journals The adhesion plaque protein, talin, is phosphorylated in vivo in chicken embryo fibroblasts exposed to a tumor-promoting phorbol ester.

1990 ◽  
Vol 1 (2) ◽  
pp. 227-236 ◽  
Author(s):  
M C Beckerle
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Chicken Embryo ◽  
Phorbol Esters ◽  
Tumor Promoter ◽  
Kinase C ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Talin is a high molecular weight phosphoprotein that is localized at adhesion plaques. We have found that talin phosphorylation increases 3.0-fold upon exposure of chicken embryo fibroblasts to the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. Talin isolated from tumor promoter-treated cells is phosphorylated on serine and threonine residues. Vinculin, a 130 kDa talin-binding protein, also exhibits increased phosphorylation in vivo in response to tumor promoter, but to a lesser degree than does talin. Because tumor-promoting phorbol esters augment protein kinase C activity, we have compared the ability of purified protein kinase C to phosphorylate talin and vinculin in vitro. Both talin and vinculin were found to be substrates for protein kinase C; however, talin was phosphorylated to a greater extent than was vinculin. Cleavage of protein kinase C-phosphorylated talin by the calcium-dependent protease (Type II) revealed that while both the resulting 190-200 and 46 kDa proteolytic peptides were phosphorylated, the majority of label was contained within the 46-kDa fragment. Although incubation of chicken embryo fibroblasts with tumor-promoting phorbol ester induces a dramatic increase in talin phosphorylation, we detected no change in the organization of stress fibers and focal contacts in these cells. Exposure of the cells to tumor promoter did, however, result in a loss of actin and talin-rich cell surface elaborations that resemble focal contact precursor structures.

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