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. 1992 Jul 2;118(2):321–333. doi: 10.1083/jcb.118.2.321

Association of p60c-src with endosomal membranes in mammalian fibroblasts

PMCID: PMC2290043  PMID: 1378446

Abstract

We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three- dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI- MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking.

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Selected References

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  1. Agard D. A., Hiraoka Y., Shaw P., Sedat J. W. Fluorescence microscopy in three dimensions. Methods Cell Biol. 1989;30:353–377. doi: 10.1016/s0091-679x(08)60986-3. [DOI] [PubMed] [Google Scholar]
  2. Aoki D., Appert H. E., Johnson D., Wong S. S., Fukuda M. N. Analysis of the substrate binding sites of human galactosyltransferase by protein engineering. EMBO J. 1990 Oct;9(10):3171–3178. doi: 10.1002/j.1460-2075.1990.tb07515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buss J. E., Kamps M. P., Gould K., Sefton B. M. The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity. J Virol. 1986 May;58(2):468–474. doi: 10.1128/jvi.58.2.468-474.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Chackalaparampil I., Shalloway D. Altered phosphorylation and activation of pp60c-src during fibroblast mitosis. Cell. 1988 Mar 25;52(6):801–810. doi: 10.1016/0092-8674(88)90422-9. [DOI] [PubMed] [Google Scholar]
  7. Chege N. W., Pfeffer S. R. Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi cisternae from the trans-Golgi network. J Cell Biol. 1990 Sep;111(3):893–899. doi: 10.1083/jcb.111.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cotton P. C., Brugge J. S. Neural tissues express high levels of the cellular src gene product pp60c-src. Mol Cell Biol. 1983 Jun;3(6):1157–1162. doi: 10.1128/mcb.3.6.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Courtneidge S. A., Levinson A. D., Bishop J. M. The protein encoded by the transforming gene of avian sarcoma virus (pp60src) and a homologous protein in normal cells (pp60proto-src) are associated with the plasma membrane. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3783–3787. doi: 10.1073/pnas.77.7.3783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Courtoy P. J., Quintart J., Baudhuin P. Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles. J Cell Biol. 1984 Mar;98(3):870–876. doi: 10.1083/jcb.98.3.870. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. David-Pfeuty T., Nouvian-Dooghe Y. Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells. J Cell Biol. 1990 Dec;111(6 Pt 2):3097–3116. doi: 10.1083/jcb.111.6.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. De Brabander M., Nuydens R., Geerts H., Hopkins C. R. Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy. Cell Motil Cytoskeleton. 1988;9(1):30–47. doi: 10.1002/cm.970090105. [DOI] [PubMed] [Google Scholar]
  13. Georgi A., Mottola-Hartshorn C., Warner A., Fields B., Chen L. B. Detection of individual fluorescently labeled reovirions in living cells. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6579–6583. doi: 10.1073/pnas.87.17.6579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Grandori C., Hanafusa H. p60c-src is complexed with a cellular protein in subcellular compartments involved in exocytosis. J Cell Biol. 1988 Dec;107(6 Pt 1):2125–2135. doi: 10.1083/jcb.107.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hamaguchi M., Hanafusa H. Association of p60src with Triton X-100-resistant cellular structure correlates with morphological transformation. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2312–2316. doi: 10.1073/pnas.84.8.2312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Henderson D., Rohrschneider L. Cytoskeletal association of pp60src. The transforming protein of the Rous sarcoma virus. Exp Cell Res. 1987 Feb;168(2):411–421. doi: 10.1016/0014-4827(87)90013-9. [DOI] [PubMed] [Google Scholar]
  18. Hiraoka Y., Agard D. A., Sedat J. W. Temporal and spatial coordination of chromosome movement, spindle formation, and nuclear envelope breakdown during prometaphase in Drosophila melanogaster embryos. J Cell Biol. 1990 Dec;111(6 Pt 2):2815–2828. doi: 10.1083/jcb.111.6.2815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hiraoka Y., Swedlow J. R., Paddy M. R., Agard D. A., Sedat J. W. Three-dimensional multiple-wavelength fluorescence microscopy for the structural analysis of biological phenomena. Semin Cell Biol. 1991 Jun;2(3):153–165. [PubMed] [Google Scholar]
  20. Johnston P. A., Cameron P. L., Stukenbrok H., Jahn R., De Camilli P., Südhof T. C. Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells. EMBO J. 1989 Oct;8(10):2863–2872. doi: 10.1002/j.1460-2075.1989.tb08434.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kaplan J. M., Varmus H. E., Bishop J. M. The src protein contains multiple domains for specific attachment to membranes. Mol Cell Biol. 1990 Mar;10(3):1000–1009. doi: 10.1128/mcb.10.3.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lewis V., Green S. A., Marsh M., Vihko P., Helenius A., Mellman I. Glycoproteins of the lysosomal membrane. J Cell Biol. 1985 Jun;100(6):1839–1847. doi: 10.1083/jcb.100.6.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Linstedt A. D., Vetter M. L., Bishop J. M., Kelly R. B. Specific association of the proto-oncogene product pp60c-src with an intracellular organelle, the PC12 synaptic vesicle. J Cell Biol. 1992 Jun;117(5):1077–1084. doi: 10.1083/jcb.117.5.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  26. Lippincott-Schwartz J., Yuan L. C., Bonifacino J. S., Klausner R. D. Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER. Cell. 1989 Mar 10;56(5):801–813. doi: 10.1016/0092-8674(89)90685-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lucocq J. M., Warren G. Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells. EMBO J. 1987 Nov;6(11):3239–3246. doi: 10.1002/j.1460-2075.1987.tb02641.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matteoni R., Kreis T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol. 1987 Sep;105(3):1253–1265. doi: 10.1083/jcb.105.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Messner D. J., Griffiths G., Kornfeld S. Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors. J Cell Biol. 1989 Jun;108(6):2149–2162. doi: 10.1083/jcb.108.6.2149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morgan D. O., Kaplan J. M., Bishop J. M., Varmus H. E. Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase. Cell. 1989 Jun 2;57(5):775–786. doi: 10.1016/0092-8674(89)90792-7. [DOI] [PubMed] [Google Scholar]
  32. Morgan D. O., Kaplan J. M., Bishop J. M., Varmus H. E. Production of p60c-src by baculovirus expression and immunoaffinity purification. Methods Enzymol. 1991;200:645–660. doi: 10.1016/0076-6879(91)00177-x. [DOI] [PubMed] [Google Scholar]
  33. Moskalewski S., Thyberg J. Disorganization and reorganization of the Golgi complex and the lysosomal system in association with mitosis. J Submicrosc Cytol Pathol. 1990 Apr;22(2):159–171. [PubMed] [Google Scholar]
  34. Paddy M. R., Belmont A. S., Saumweber H., Agard D. A., Sedat J. W. Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery. Cell. 1990 Jul 13;62(1):89–106. doi: 10.1016/0092-8674(90)90243-8. [DOI] [PubMed] [Google Scholar]
  35. Pool R. R., Jr, Maurey K. M., Storrie B. Characterization of pinocytic vesicles from CHO cells: resolution of pinosomes from lysosomes by analytical centrifugation. Cell Biol Int Rep. 1983 May;7(5):361–367. doi: 10.1016/0309-1651(83)90076-0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]
  39. Rohrschneider L. R. Immunofluorescence on avian sarcoma virus-transformed cells: localization of the src gene product. Cell. 1979 Jan;16(1):11–24. doi: 10.1016/0092-8674(79)90183-1. [DOI] [PubMed] [Google Scholar]
  40. Soriano P., Montgomery C., Geske R., Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. doi: 10.1016/0092-8674(91)90499-o. [DOI] [PubMed] [Google Scholar]
  41. Stoorvogel W., Geuze H. J., Griffith J. M., Schwartz A. L., Strous G. J. Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells. J Cell Biol. 1989 Jun;108(6):2137–2148. doi: 10.1083/jcb.108.6.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stoorvogel W., Strous G. J., Geuze H. J., Oorschot V., Schwartz A. L. Late endosomes derive from early endosomes by maturation. Cell. 1991 May 3;65(3):417–427. doi: 10.1016/0092-8674(91)90459-c. [DOI] [PubMed] [Google Scholar]
  43. Thompson J. A., Lau A. L., Cunningham D. D. Selective radiolabeling of cell surface proteins to a high specific activity. Biochemistry. 1987 Feb 10;26(3):743–750. doi: 10.1021/bi00377a014. [DOI] [PubMed] [Google Scholar]
  44. Tuomikoski T., Felix M. A., Dorée M., Gruenberg J. Inhibition of endocytic vesicle fusion in vitro by the cell-cycle control protein kinase cdc2. Nature. 1989 Dec 21;342(6252):942–945. doi: 10.1038/342942a0. [DOI] [PubMed] [Google Scholar]
  45. Willingham M. C., Jay G., Pastan I. Localization of the ASV src gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1979 Sep;18(1):125–134. doi: 10.1016/0092-8674(79)90361-1. [DOI] [PubMed] [Google Scholar]
  46. Wood S. A., Park J. E., Brown W. J. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell. 1991 Nov 1;67(3):591–600. doi: 10.1016/0092-8674(91)90533-5. [DOI] [PubMed] [Google Scholar]
  47. Woodman P. G., Mundy D. I., Cohen P., Warren G. Cell-free fusion of endocytic vesicles is regulated by phosphorylation. J Cell Biol. 1992 Jan;116(2):331–338. doi: 10.1083/jcb.116.2.331. [DOI] [PMC free article] [PubMed] [Google Scholar]

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