Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 Nov;83(21):8191–8195. doi: 10.1073/pnas.83.21.8191

Protein tyrosine phosphorylation in the cell cycle of BALB/c 3T3 fibroblasts.

A O Morla, J Y Wang
PMCID: PMC386893  PMID: 3464948

Abstract

Cell cycle-dependent regulation of protein tyrosine phosphorylation in normal BALB/c 3T3 fibroblasts was examined by immunoblotting with a high-affinity antibody specific for phosphotyrosine. At least 15 different tyrosine-phosphorylated proteins are found in normal 3T3 cells. The level of tyrosine phosphorylation is higher in growing cells than in quiescent cells. However, a prominent tyrosine-phosphorylated protein of Mr 150,000 is present in quiescent cells, and its level is inversely proportional to the growth rate of these fibroblasts. Stimulation of quiescent cells with serum causes a major, yet transient, increase in tyrosine phosphorylation. The immediate tyrosine phosphorylation reactions in response to serum stimulation are independent of protein synthesis, but tyrosine phosphorylation reactions occurring later in the G1 phase of the cell cycle are inhibited by cycloheximide. Thus, tyrosine phosphorylation of proteins in normal 3T3 cells occurs predominantly at the G0 to G1 transition of the cell cycle. Maintenance of steady-state tyrosine phosphorylation is dependent on the presence of serum, but at least one tyrosine phosphorylation reaction occurs in the absence of cell growth.

Full text

PDF
8191

Images in this article

8192Image
on p.8192
8192Image
on p.8192
8192Image
on p.8192
8192Image
on p.8192
8193Image
on p.8193
8193Image
on p.8193
8193Image
on p.8193

Selected References

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

  1. Antoniades H. N., Scher C. D., Stiles C. D. Purification of human platelet-derived growth factor. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1809–1813. doi: 10.1073/pnas.76.4.1809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bishop J. M. Cellular oncogenes and retroviruses. Annu Rev Biochem. 1983;52:301–354. doi: 10.1146/annurev.bi.52.070183.001505. [DOI] [PubMed] [Google Scholar]
  3. Brugge J. S., Cotton P. C., Queral A. E., Barrett J. N., Nonner D., Keane R. W. Neurones express high levels of a structurally modified, activated form of pp60c-src. Nature. 1985 Aug 8;316(6028):554–557. doi: 10.1038/316554a0. [DOI] [PubMed] [Google Scholar]
  4. Campisi J., Pardee A. B. Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor. Mol Cell Biol. 1984 Sep;4(9):1807–1814. doi: 10.1128/mcb.4.9.1807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carpenter G., Cohen S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol. 1976 Oct;71(1):159–171. doi: 10.1083/jcb.71.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen S., Carpenter G., King L., Jr Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J Biol Chem. 1980 May 25;255(10):4834–4842. [PubMed] [Google Scholar]
  7. Cooper J. A., Bowen-Pope D. F., Raines E., Ross R., Hunter T. Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins. Cell. 1982 Nov;31(1):263–273. doi: 10.1016/0092-8674(82)90426-3. [DOI] [PubMed] [Google Scholar]
  8. Cooper J. A., Hunter T. Four different classes of retroviruses induce phosphorylation of tyrosines present in similar cellular proteins. Mol Cell Biol. 1981 May;1(5):394–407. doi: 10.1128/mcb.1.5.394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooper J. A., Hunter T. Regulation of cell growth and transformation by tyrosine-specific protein kinases: the search for important cellular substrate proteins. Curr Top Microbiol Immunol. 1983;107:125–161. doi: 10.1007/978-3-642-69075-4_4. [DOI] [PubMed] [Google Scholar]
  10. Daniel T. O., Tremble P. M., Frackelton A. R., Jr, Williams L. T. Purification of the platelet-derived growth factor receptor by using an anti-phosphotyrosine antibody. Proc Natl Acad Sci U S A. 1985 May;82(9):2684–2687. doi: 10.1073/pnas.82.9.2684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ek B., Heldin C. H. Use of an antiserum against phosphotyrosine for the identification of phosphorylated components in human fibroblasts stimulated by platelet-derived growth factor. J Biol Chem. 1984 Sep 10;259(17):11145–11152. [PubMed] [Google Scholar]
  12. Ek B., Westermark B., Wasteson A., Heldin C. H. Stimulation of tyrosine-specific phosphorylation by platelet-derived growth factor. Nature. 1982 Feb 4;295(5848):419–420. doi: 10.1038/295419a0. [DOI] [PubMed] [Google Scholar]
  13. Foulkes J. G., Chow M., Gorka C., Frackelton A. R., Jr, Baltimore D. Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus. J Biol Chem. 1985 Jul 5;260(13):8070–8077. [PubMed] [Google Scholar]
  14. Frackelton A. R., Jr, Ross A. H., Eisen H. N. Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells. Mol Cell Biol. 1983 Aug;3(8):1343–1352. doi: 10.1128/mcb.3.8.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frackelton A. R., Jr, Tremble P. M., Williams L. T. Evidence for the platelet-derived growth factor-stimulated tyrosine phosphorylation of the platelet-derived growth factor receptor in vivo. Immunopurification using a monoclonal antibody to phosphotyrosine. J Biol Chem. 1984 Jun 25;259(12):7909–7915. [PubMed] [Google Scholar]
  16. Glenn K., Bowen-Pope D. F., Ross R. Platelet-derived growth factor. III. Identification of a platelet-derived growth factor receptor by affinity labeling. J Biol Chem. 1982 May 10;257(9):5172–5176. [PubMed] [Google Scholar]
  17. Golden A., Nemeth S. P., Brugge J. S. Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity. Proc Natl Acad Sci U S A. 1986 Feb;83(4):852–856. doi: 10.1073/pnas.83.4.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gorden P., Carpentier J. L., Cohen S., Orci L. Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc Natl Acad Sci U S A. 1978 Oct;75(10):5025–5029. doi: 10.1073/pnas.75.10.5025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heldin C. H., Ek B., Rönnstrand L. Characterization of the receptor for platelet-derived growth factor on human fibroblasts. Demonstration of an intimate relationship with a 185,000-Dalton substrate for the platelet-derived growth factor-stimulated kinase. J Biol Chem. 1983 Aug 25;258(16):10054–10061. [PubMed] [Google Scholar]
  20. Hultquist D. E., Moyer R. W., Boyer P. D. The preparation and characterization of 1-phosphohistidine and 3-phosphohistidine. Biochemistry. 1966 Jan;5(1):322–331. doi: 10.1021/bi00865a041. [DOI] [PubMed] [Google Scholar]
  21. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  22. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jacobs S., Kull F. C., Jr, Earp H. S., Svoboda M. E., Van Wyk J. J., Cuatrecasas P. Somatomedin-C stimulates the phosphorylation of the beta-subunit of its own receptor. J Biol Chem. 1983 Aug 25;258(16):9581–9584. [PubMed] [Google Scholar]
  24. Knutson V. P., Ronnett G. V., Lane M. D. The effects of cycloheximide and chloroquine on insulin receptor metabolism. Differential effects on receptor recycling and inactivation and insulin degradation. J Biol Chem. 1985 Nov 15;260(26):14180–14188. [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Maher P. A., Pasquale E. B., Wang J. Y., Singer S. J. Phosphotyrosine-containing proteins are concentrated in focal adhesions and intercellular junctions in normal cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6576–6580. doi: 10.1073/pnas.82.19.6576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ross A. H., Baltimore D., Eisen H. N. Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature. 1981 Dec 17;294(5842):654–656. doi: 10.1038/294654a0. [DOI] [PubMed] [Google Scholar]
  28. Scher C. D., Shepard R. C., Antoniades H. N., Stiles C. D. Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim Biophys Acta. 1979 Aug 10;560(2):217–241. doi: 10.1016/0304-419x(79)90020-9. [DOI] [PubMed] [Google Scholar]
  29. Sefton B. M., Hunter T., Beemon K., Eckhart W. Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus. Cell. 1980 Jul;20(3):807–816. doi: 10.1016/0092-8674(80)90327-x. [DOI] [PubMed] [Google Scholar]
  30. Sherr C. J., Rettenmier C. W., Sacca R., Roussel M. F., Look A. T., Stanley E. R. The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell. 1985 Jul;41(3):665–676. doi: 10.1016/s0092-8674(85)80047-7. [DOI] [PubMed] [Google Scholar]
  31. Sorge L. K., Levy B. T., Maness P. F. pp60c-src is developmentally regulated in the neural retina. Cell. 1984 Feb;36(2):249–257. doi: 10.1016/0092-8674(84)90218-6. [DOI] [PubMed] [Google Scholar]
  32. Wang J. Y., Baltimore D. Localization of tyrosine kinase-coding region in v-abl oncogene by the expression of v-abl-encoded proteins in bacteria. J Biol Chem. 1985 Jan 10;260(1):64–71. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES