Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 Nov;5(11):3097–3107. doi: 10.1128/mcb.5.11.3097

Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants.

L Rohrschneider, S Reynolds
PMCID: PMC369124  PMID: 3018500

Abstract

We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology.

Full text

PDF
3097

Images in this article

3099Image
on p.3099
3100Image
on p.3100
3102Image
on p.3102
3103Image
on p.3103
3104Image
on p.3104
3105Image
on p.3105
3105Image
on p.3105

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams S. L., Sobel M. E., Howard B. H., Olden K., Yamada K. M., de Crombrugghe B., Pastan I. Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3399–3403. doi: 10.1073/pnas.74.8.3399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ali I. U., Mautner V., Lanza R., Hynes R. O. Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein. Cell. 1977 May;11(1):115–126. doi: 10.1016/0092-8674(77)90322-1. [DOI] [PubMed] [Google Scholar]
  3. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson D. D., Beckmann R. P., Harms E. H., Nakamura K., Weber M. J. Biological properties of "partial" transformation mutants of Rous sarcoma virus and characterization of their pp60src kinase. J Virol. 1981 Jan;37(1):445–458. doi: 10.1128/jvi.37.1.445-458.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Antler A. M., Greenberg M. E., Edelman G. M., Hanafusa H. Increased phosphorylation of tyrosine in vinculin does not occur upon transformation by some avian sarcoma viruses. Mol Cell Biol. 1985 Jan;5(1):263–267. doi: 10.1128/mcb.5.1.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Asch B. B., Kamat B. R., Burstein N. A. Interactions of normal, dysplastic, and malignant mammary epithelial cells with fibronectin in vivo and in vitro. Cancer Res. 1981 Jun;41(6):2115–2125. [PubMed] [Google Scholar]
  7. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  8. Buss J. E., Sefton B. M. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog. J Virol. 1985 Jan;53(1):7–12. doi: 10.1128/jvi.53.1.7-12.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen W. T., Hasegawa E., Hasegawa T., Weinstock C., Yamada K. M. Development of cell surface linkage complexes in cultured fibroblasts. J Cell Biol. 1985 Apr;100(4):1103–1114. doi: 10.1083/jcb.100.4.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen W. T., Olden K., Bernard B. A., Chu F. F. Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites. J Cell Biol. 1984 Apr;98(4):1546–1555. doi: 10.1083/jcb.98.4.1546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cross F. R., Garber E. A., Pellman D., Hanafusa H. A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation. Mol Cell Biol. 1984 Sep;4(9):1834–1842. doi: 10.1128/mcb.4.9.1834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Damsky C. H., Knudsen K. A., Bradley D., Buck C. A., Horwitz A. F. Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture. J Cell Biol. 1985 May;100(5):1528–1539. doi: 10.1083/jcb.100.5.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fujita D. J., Bechberger J., Nedic I. Four Rous sarcoma virus mutants which affect transformed cell morphology exhibit altered src gene products. Virology. 1981 Oct 15;114(1):256–260. doi: 10.1016/0042-6822(81)90272-5. [DOI] [PubMed] [Google Scholar]
  14. Fujita D. J., Boschek C. B., Ziemiecki A., Friis R. R. An avian sarcoma virus mutant which produces an aberrant transformation affecting cell morphology. Virology. 1981 May;111(1):223–238. doi: 10.1016/0042-6822(81)90667-x. [DOI] [PubMed] [Google Scholar]
  15. Garber E. A., Krueger J. G., Hanafusa H., Goldberg A. R. Only membrane-associated RSV src proteins have amino-terminally bound lipid. Nature. 1983 Mar 10;302(5904):161–163. doi: 10.1038/302161a0. [DOI] [PubMed] [Google Scholar]
  16. Gentry L. E., Rohrschneider L. R., Casnellie J. E., Krebs E. G. Antibodies to a defined region of pp60src neutralize the tyrosine-specific kinase activity. J Biol Chem. 1983 Sep 25;258(18):11219–11228. [PubMed] [Google Scholar]
  17. Hirano H., Yamada Y., Sullivan M., de Crombrugghe B., Pastan I., Yamada K. M. Isolation of genomic DNA clones spanning the entire fibronectin gene. Proc Natl Acad Sci U S A. 1983 Jan;80(1):46–50. doi: 10.1073/pnas.80.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hsieh P., Chen L. B. Behavior of cells seeded in isolated fibronectin matrices. J Cell Biol. 1983 May;96(5):1208–1217. doi: 10.1083/jcb.96.5.1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hughes S., Mellstrom K., Kosik E., Tamanoi F., Brugge J. Mutation of a termination codon affects src initiation. Mol Cell Biol. 1984 Sep;4(9):1738–1746. doi: 10.1128/mcb.4.9.1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iwashita S., Kitamura N., Yoshida M. Molecular events leading to fusiform morphological transformation by partial src deletion mutant of Rous sarcoma virus. Virology. 1983 Mar;125(2):419–431. doi: 10.1016/0042-6822(83)90213-1. [DOI] [PubMed] [Google Scholar]
  21. Kahn P., Nakamura K., Shin S., Smith R. E., Weber M. J. Tumorigenicity of partial transformation mutants of Rous sarcoma virus. J Virol. 1982 May;42(2):602–611. doi: 10.1128/jvi.42.2.602-611.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kitamura N., Yoshida M. Small deletion in src of Rous sarcoma virus modifying transformation phenotypes: identification of 207-nucleotide deletion and its smaller product with protein kinase activity. J Virol. 1983 Jun;46(3):985–992. doi: 10.1128/jvi.46.3.985-992.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Krueger J. G., Garber E. A., Chin S. S., Hanafusa H., Goldberg A. R. Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation. Mol Cell Biol. 1984 Mar;4(3):454–467. doi: 10.1128/mcb.4.3.454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nigg E. A., Sefton B. M., Hunter T., Walter G., Singer S. J. Immunofluorescent localization of the transforming protein of Rous sarcoma virus with antibodies against a synthetic src peptide. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5322–5326. doi: 10.1073/pnas.79.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Olden K., Yamada K. M. Mechanism of the decrease in the major cell surface protein of chick embryo fibroblasts after transformation. Cell. 1977 Aug;11(4):957–969. doi: 10.1016/0092-8674(77)90307-5. [DOI] [PubMed] [Google Scholar]
  27. Oppermann H., Levinson A. D., Varmus H. E. The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the sec gene of avian sarcoma virus. Virology. 1981 Jan 15;108(1):47–70. doi: 10.1016/0042-6822(81)90526-2. [DOI] [PubMed] [Google Scholar]
  28. Parry G., Soo W. J., Bissell M. J. The uncoupled regulation of fibronectin and collagen synthesis in Rous sarcoma virus transformed avian tendon cells. J Biol Chem. 1979 Dec 10;254(23):11763–11766. [PubMed] [Google Scholar]
  29. Rohrschneider L. R. Adhesion plaques of Rous sarcoma virus-transformed cells contain the src gene product. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3514–3518. doi: 10.1073/pnas.77.6.3514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rohrschneider L., Rosok M. J. Transformation parameters and pp60src localization in cells infected with partial transformation mutants of Rous sarcoma virus. Mol Cell Biol. 1983 Apr;3(4):731–746. doi: 10.1128/mcb.3.4.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  32. Shriver K., Rohrschneider L. Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells. J Cell Biol. 1981 Jun;89(3):525–535. doi: 10.1083/jcb.89.3.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Singer I. I., Paradiso P. R. A transmembrane relationship between fibronectin and vinculin (130 kd protein): serum modulation in normal and transformed hamster fibroblasts. Cell. 1981 May;24(2):481–492. doi: 10.1016/0092-8674(81)90339-1. [DOI] [PubMed] [Google Scholar]
  34. Singer I. I. The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts. Cell. 1979 Mar;16(3):675–685. doi: 10.1016/0092-8674(79)90040-0. [DOI] [PubMed] [Google Scholar]
  35. Smith H. S., Riggs J. L., Mosesson M. W. Production of fibronectin by human epithelial cells in culture. Cancer Res. 1979 Oct;39(10):4138–4144. [PubMed] [Google Scholar]
  36. TEMIN H. M. The control of cellular morphology in embryonic cells infected with rous sarcoma virus in vitro. Virology. 1960 Feb;10:182–197. doi: 10.1016/0042-6822(60)90038-6. [DOI] [PubMed] [Google Scholar]
  37. Varmus H. E., Quintrell N., Wyke J. Revertants of an ASV-transformed rat cell line have lost the complete provius or sustained mutations in src. Virology. 1981 Jan 15;108(1):28–46. doi: 10.1016/0042-6822(81)90525-0. [DOI] [PubMed] [Google Scholar]
  38. Wagner D. D., Ivatt R., Destree A. T., Hynes R. O. Similarities and differences between the fibronectins of normal and transformed hamster cells. J Biol Chem. 1981 Nov 25;256(22):11708–11715. [PubMed] [Google Scholar]
  39. Yamada K. M., Yamada S. S., Pastan I. Cell surface protein partially restores morphology, adhesiveness, and contact inhibition of movement to transformed fibroblasts. Proc Natl Acad Sci U S A. 1976 Apr;73(4):1217–1221. doi: 10.1073/pnas.73.4.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yoshii S., Vogt P. K. A mutant of rous sarcoma virus (type O) causing fusiform cell transformation. Proc Soc Exp Biol Med. 1970 Nov;135(2):297–301. doi: 10.3181/00379727-135-35039. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES