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. 1993 Aug 2;122(4):903–914. doi: 10.1083/jcb.122.4.903

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype

PMCID: PMC2119593  PMID: 7688751

Abstract

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, becomes upregulated during cell proliferation and transformation. Here we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor alpha-difluoromethyl ornithine (DFMO) to the cells (in polyamine-free medium) before shift to permissive temperature prevented the depolymerization of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro tyrosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed in the tyrosine phosphorylation of phosphatidylinositol 3-kinase and proteins of 33 and 36 kD upon the temperature shift, and these hyperphosphorylations were partially inhibited by DFMO. A DFMO-sensitive increase was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-containing proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to coimmunoprecipitate with pp60v-src in a transformation-related manner. Altogether, this model offers a tool for sorting out the protein phosphorylations and associations critical for the transformed phenotype triggered by pp60v- src, and implicates a pivotal role for polyamines in cell transformation.

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Selected References

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  1. Anderson D., Koch C. A., Grey L., Ellis C., Moran M. F., Pawson T. Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. Science. 1990 Nov 16;250(4983):979–982. doi: 10.1126/science.2173144. [DOI] [PubMed] [Google Scholar]
  2. Auvinen M., Paasinen A., Andersson L. C., Hölttä E. Ornithine decarboxylase activity is critical for cell transformation. Nature. 1992 Nov 26;360(6402):355–358. doi: 10.1038/360355a0. [DOI] [PubMed] [Google Scholar]
  3. Bachrach U., Shtorch A. Formation of cadaverine as an effect of alpha-difluoromethylornithine on chick embryo fibroblasts transformed by rous sarcoma virus. Cancer Res. 1985 May;45(5):2159–2164. [PubMed] [Google Scholar]
  4. Balasundaram D., Tabor C. W., Tabor H. Spermidine or spermine is essential for the aerobic growth of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5872–5876. doi: 10.1073/pnas.88.13.5872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boschek C. B., Jockusch B. M., Friis R. R., Back R., Grundmann E., Bauer H. Early changes in the distribution and organization of microfilament proteins during cell transformation. Cell. 1981 Apr;24(1):175–184. doi: 10.1016/0092-8674(81)90513-4. [DOI] [PubMed] [Google Scholar]
  6. Bouton A. H., Kanner S. B., Vines R. R., Wang H. C., Gibbs J. B., Parsons J. T. Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein. Mol Cell Biol. 1991 Feb;11(2):945–953. doi: 10.1128/mcb.11.2.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brady G., Williams R. L., Nordström K., Vennström B., Iscove N. N. A selectable temperature-sensitive v-src Moloney retrovirus. Oncogene. 1988 Dec;3(6):687–689. [PubMed] [Google Scholar]
  8. Brott B. K., Decker S., O'Brien M. C., Jove R. Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein. Mol Cell Biol. 1991 Oct;11(10):5059–5067. doi: 10.1128/mcb.11.10.5059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brott B. K., Decker S., Shafer J., Gibbs J. B., Jove R. GTPase-activating protein interactions with the viral and cellular Src kinases. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):755–759. doi: 10.1073/pnas.88.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  11. Crompton M. R., Moss S. E., Crumpton M. J. Diversity in the lipocortin/calpactin family. Cell. 1988 Oct 7;55(1):1–3. doi: 10.1016/0092-8674(88)90002-5. [DOI] [PubMed] [Google Scholar]
  12. Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
  13. Drubin D. G., Mulholland J., Zhu Z. M., Botstein D. Homology of a yeast actin-binding protein to signal transduction proteins and myosin-I. Nature. 1990 Jan 18;343(6255):288–290. doi: 10.1038/343288a0. [DOI] [PubMed] [Google Scholar]
  14. Eberle M., Traynor-Kaplan A. E., Sklar L. A., Norgauer J. Is there a relationship between phosphatidylinositol trisphosphate and F-actin polymerization in human neutrophils? J Biol Chem. 1990 Oct 5;265(28):16725–16728. [PubMed] [Google Scholar]
  15. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  16. Erikson R. L., Purchio A. F., Erikson E., Collett M. S., Brugge J. S. Molecular events in cells transformed by Rous Sarcoma virus. J Cell Biol. 1980 Nov;87(2 Pt 1):319–325. doi: 10.1083/jcb.87.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Escobedo J. A., Kaplan D. R., Kavanaugh W. M., Turck C. W., Williams L. T. A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. Mol Cell Biol. 1991 Feb;11(2):1125–1132. doi: 10.1128/mcb.11.2.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fukui Y., Hanafusa H. Phosphatidylinositol kinase activity associates with viral p60src protein. Mol Cell Biol. 1989 Apr;9(4):1651–1658. doi: 10.1128/mcb.9.4.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fukui Y., O'Brien M. C., Hanafusa H. Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix. Mol Cell Biol. 1991 Mar;11(3):1207–1213. doi: 10.1128/mcb.11.3.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gazdar A. F., Stull H. B., Kilton L. J., Bachrach U. Increased ornithine decarboxylase activity in murine sarcoma virus infected cells. Nature. 1976 Aug 19;262(5570):696–698. doi: 10.1038/262696a0. [DOI] [PubMed] [Google Scholar]
  21. Gilmour S. K., Verma A. K., Madara T., O'Brien T. G. Regulation of ornithine decarboxylase gene expression in mouse epidermis and epidermal tumors during two-stage tumorigenesis. Cancer Res. 1987 Mar 1;47(5):1221–1225. [PubMed] [Google Scholar]
  22. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gu M. X., York J. D., Warshawsky I., Majerus P. W. Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein 4.1. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5867–5871. doi: 10.1073/pnas.88.13.5867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Haddox M. K., Magun B. E., Russell D. H. Ornithine decarboxylase induction during B1 progression of normal and Rous sarcoma virus-transformed cells. Cancer Res. 1980 Mar;40(3):604–608. [PubMed] [Google Scholar]
  25. Hall A. ras and GAP--who's controlling whom? Cell. 1990 Jun 15;61(6):921–923. doi: 10.1016/0092-8674(90)90054-i. [DOI] [PubMed] [Google Scholar]
  26. Hamaguchi M., Matsuda M., Hanafusa H. A glycoprotein in the plasma membrane matrix as a major potential substrate of p60v-src. Mol Cell Biol. 1990 Feb;10(2):830–836. doi: 10.1128/mcb.10.2.830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Heby O., Persson L. Molecular genetics of polyamine synthesis in eukaryotic cells. Trends Biochem Sci. 1990 Apr;15(4):153–158. doi: 10.1016/0968-0004(90)90216-x. [DOI] [PubMed] [Google Scholar]
  28. Hirai H., Varmus H. E. Mutations in src homology regions 2 and 3 of activated chicken c-src that result in preferential transformation of mouse or chicken cells. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8592–8596. doi: 10.1073/pnas.87.21.8592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hölttä E., Hovi T. Polyamine depletion results in impairment of polyribosome formation and protein synthesis before onset of DNA synthesis in mitogen-activated human lymphocytes. Eur J Biochem. 1985 Oct 1;152(1):229–237. doi: 10.1111/j.1432-1033.1985.tb09188.x. [DOI] [PubMed] [Google Scholar]
  30. Hölttä E., Sistonen L., Alitalo K. The mechanisms of ornithine decarboxylase deregulation in c-Ha-ras oncogene-transformed NIH 3T3 cells. J Biol Chem. 1988 Mar 25;263(9):4500–4507. [PubMed] [Google Scholar]
  31. Isacke C. M., Lindberg R. A., Hunter T. Synthesis of p36 and p35 is increased when U-937 cells differentiate in culture but expression is not inducible by glucocorticoids. Mol Cell Biol. 1989 Jan;9(1):232–240. doi: 10.1128/mcb.9.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jakobovits E. B., Majors J. E., Varmus H. E. Hormonal regulation of the Rous sarcoma virus src gene via a heterologous promoter defines a threshold dose for cellular transformation. Cell. 1984 Oct;38(3):757–765. doi: 10.1016/0092-8674(84)90271-x. [DOI] [PubMed] [Google Scholar]
  33. Kamps M. P., Buss J. E., Sefton B. M. Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation. Cell. 1986 Apr 11;45(1):105–112. doi: 10.1016/0092-8674(86)90542-8. [DOI] [PubMed] [Google Scholar]
  34. Kamps M. P., Sefton B. M. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [PubMed] [Google Scholar]
  35. Kanner S. B., Reynolds A. B., Wang H. C., Vines R. R., Parsons J. T. The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J. 1991 Jul;10(7):1689–1698. doi: 10.1002/j.1460-2075.1991.tb07693.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kozma L. M., Reynolds A. B., Weber M. J. Glycoprotein tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryo fibroblasts. Mol Cell Biol. 1990 Feb;10(2):837–841. doi: 10.1128/mcb.10.2.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kypta R. M., Goldberg Y., Ulug E. T., Courtneidge S. A. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell. 1990 Aug 10;62(3):481–492. doi: 10.1016/0092-8674(90)90013-5. [DOI] [PubMed] [Google Scholar]
  38. Ling L. E., Druker B. J., Cantley L. C., Roberts T. M. Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells. J Virol. 1992 Mar;66(3):1702–1708. doi: 10.1128/jvi.66.3.1702-1708.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Melamed I., Downey G. P., Roifman C. M. Tyrosine phosphorylation is essential for microfilament assembly in B lymphocytes. Biochem Biophys Res Commun. 1991 May 15;176(3):1424–1429. doi: 10.1016/0006-291x(91)90445-d. [DOI] [PubMed] [Google Scholar]
  40. Moran M. F., Koch C. A., Anderson D., Ellis C., England L., Martin G. S., Pawson T. Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8622–8626. doi: 10.1073/pnas.87.21.8622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Moran M. F., Polakis P., McCormick F., Pawson T., Ellis C. Protein-tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution, and activity of p21ras GTPase-activating protein. Mol Cell Biol. 1991 Apr;11(4):1804–1812. doi: 10.1128/mcb.11.4.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Parker R. C., Varmus H. E., Bishop J. M. Expression of v-src and chicken c-src in rat cells demonstrates qualitative differences between pp60v-src and pp60c-src. Cell. 1984 May;37(1):131–139. doi: 10.1016/0092-8674(84)90308-8. [DOI] [PubMed] [Google Scholar]
  43. Pegg A. E. Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res. 1988 Feb 15;48(4):759–774. [PubMed] [Google Scholar]
  44. Pohjanpelto P., Virtanen I., Hölttä E. Polyamine starvation causes disappearance of actin filaments and microtubules in polyamine-auxotrophic CHO cells. Nature. 1981 Oct 8;293(5832):475–477. doi: 10.1038/293475a0. [DOI] [PubMed] [Google Scholar]
  45. Pronk G. J., Polakis P., Wong G., de Vries-Smits A. M., Bos J. L., McCormick F. Association of a tyrosine kinase activity with GAP complexes in v-src transformed fibroblasts. Oncogene. 1992 Feb;7(2):389–394. [PubMed] [Google Scholar]
  46. Resh M. D., Erikson R. L. Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v-src and pp60c-src molecules. J Cell Biol. 1985 Feb;100(2):409–417. doi: 10.1083/jcb.100.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Resh M. D., Ling H. P. Identification of a 32K plasma membrane protein that binds to the myristylated amino-terminal sequence of p60v-src. Nature. 1990 Jul 5;346(6279):84–86. doi: 10.1038/346084a0. [DOI] [PubMed] [Google Scholar]
  48. Reynolds A. B., Roesel D. J., Kanner S. B., Parsons J. T. Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene. Mol Cell Biol. 1989 Feb;9(2):629–638. doi: 10.1128/mcb.9.2.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sartor O., Sameshima J. H., Robbins K. C. Differential association of cellular proteins with family protein-tyrosine kinases. J Biol Chem. 1991 Apr 5;266(10):6462–6466. [PubMed] [Google Scholar]
  50. Sawyer S. T., Cohen S. Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells. J Biol Chem. 1985 Jul 15;260(14):8233–8236. [PubMed] [Google Scholar]
  51. Sefton B. M., Hunter T., Ball E. H., Singer S. J. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. doi: 10.1016/0092-8674(81)90512-2. [DOI] [PubMed] [Google Scholar]
  52. Sefton B. M. The viral tyrosine protein kinases. Curr Top Microbiol Immunol. 1986;123:39–72. doi: 10.1007/978-3-642-70810-7_3. [DOI] [PubMed] [Google Scholar]
  53. Seki J., Owada M. K., Sakato N., Fujio H. Direct identification of phosphotyrosine-containing proteins in some retrovirus-transformed cells by use of anti-phosphotyrosine antibody. Cancer Res. 1986 Feb;46(2):907–916. [PubMed] [Google Scholar]
  54. Sistonen L., Hölttä E., Lehväslaiho H., Lehtola L., Alitalo K. Activation of the neu tyrosine kinase induces the fos/jun transcription factor complex, the glucose transporter and ornithine decarboxylase. J Cell Biol. 1989 Nov;109(5):1911–1919. doi: 10.1083/jcb.109.5.1911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sistonen L., Hölttä E., Mäkelä T. P., Keski-Oja J., Alitalo K. The cellular response to induction of the p21 c-Ha-ras oncoprotein includes stimulation of jun gene expression. EMBO J. 1989 Mar;8(3):815–822. doi: 10.1002/j.1460-2075.1989.tb03442.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tabor C. W., Tabor H. Polyamines. Annu Rev Biochem. 1984;53:749–790. doi: 10.1146/annurev.bi.53.070184.003533. [DOI] [PubMed] [Google Scholar]
  57. Wasilenko W. J., Payne D. M., Fitzgerald D. L., Weber M. J. Phosphorylation and activation of epidermal growth factor receptors in cells transformed by the src oncogene. Mol Cell Biol. 1991 Jan;11(1):309–321. doi: 10.1128/mcb.11.1.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wyke J. A., Beamand J. A., Varmus H. E. Factors affecting phenotypic reversion of rat cells transformed by avian sarcoma virus. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 2):1065–1075. doi: 10.1101/sqb.1980.044.01.115. [DOI] [PubMed] [Google Scholar]
  59. Yang Q., Tonks N. K. Isolation of a cDNA clone encoding a human protein-tyrosine phosphatase with homology to the cytoskeletal-associated proteins band 4.1, ezrin, and talin. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):5949–5953. doi: 10.1073/pnas.88.14.5949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zokas L., Glenney J. R., Jr The calpactin light chain is tightly linked to the cytoskeletal form of calpactin I: studies using monoclonal antibodies to calpactin subunits. J Cell Biol. 1987 Nov;105(5):2111–2121. doi: 10.1083/jcb.105.5.2111. [DOI] [PMC free article] [PubMed] [Google Scholar]

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