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. 1996 May 1;315(Pt 3):925–930. doi: 10.1042/bj3150925

Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed in a stroma-inducing clone from a mouse Lewis lung carcinoma.

PMCID: PMC1217295  PMID: 8645178

Abstract

We previously reported that a mouse Lewis lung carcinoma-derived stroma-inducing clone, P29, highly expresses a syndecan-like proteoglycan exhibiting specific binding to fibronectin, a major constituent of the interstitial matrix formed by the induced stromal cells, via its heparan sulphate chains [Itano, Oguri, Nakanishi and Okayama (1993) J. Biochem. (Tokyo) 114, 862-873]. On metabolic labelling of the proteoglycan with [32P]Pi, followed by identification of the radiolabelled material using glycanases, almost all the isotope was found to have been incorporated into a core portion of molecular mass 48 kDa, which was generated by digestion with heparan sulphate lyase I plus chondroitin ABC lyase. Immunoblotting of the core protein with a monoclonal antibody, F58-6G12, demonstrated that the proteoglycan was mouse syndecan-2. CsCl-density-gradient centrifugation after mild treatment of liposome-intercalated 32P-labelled syndecan-2 with trypsin resulted in clear separation of the radioactivity into a bottom fraction containing all the glycosaminoglycans (accounting for 40% of the total radioactivity) and a top fraction containing liposome-associated peptides (60%). The former isotope was shown to be linked covalently to both heparan sulphate and chondroitin sulphate chains, probably at their bridge regions. The latter was mostly attributed to phosphoserine, the one and only phosphorylated amino acid released on acid hydrolysis of this proteoglycan, strongly suggesting that the phosphorylation occurs at a specific serine residue(s) in the cytoplasmic domain of the core protein.

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Selected References

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  1. Bernfield M., Banerjee S. D. The turnover of basal lamina glycosaminoglycan correlates with epithelial morphogenesis. Dev Biol. 1982 Apr;90(2):291–305. doi: 10.1016/0012-1606(82)90378-5. [DOI] [PubMed] [Google Scholar]
  2. Bernfield M., Kokenyesi R., Kato M., Hinkes M. T., Spring J., Gallo R. L., Lose E. J. Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu Rev Cell Biol. 1992;8:365–393. doi: 10.1146/annurev.cb.08.110192.002053. [DOI] [PubMed] [Google Scholar]
  3. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  4. Ekblom P., Vestweber D., Kemler R. Cell-matrix interactions and cell adhesion during development. Annu Rev Cell Biol. 1986;2:27–47. doi: 10.1146/annurev.cb.02.110186.000331. [DOI] [PubMed] [Google Scholar]
  5. Esko J. D., Rostand K. S., Weinke J. L. Tumor formation dependent on proteoglycan biosynthesis. Science. 1988 Aug 26;241(4869):1092–1096. doi: 10.1126/science.3137658. [DOI] [PubMed] [Google Scholar]
  6. Feramisco J. R., Glass D. B., Krebs E. G. Optimal spatial requirements for the location of basic residues in peptide substrates for the cyclic AMP-dependent protein kinase. J Biol Chem. 1980 May 10;255(9):4240–4245. [PubMed] [Google Scholar]
  7. Fransson L. A., Silverberg I., Carlstedt I. Structure of the heparan sulfate-protein linkage region. Demonstration of the sequence galactosyl-galactosyl-xylose-2-phosphate. J Biol Chem. 1985 Nov 25;260(27):14722–14726. [PubMed] [Google Scholar]
  8. Gallagher J. T. The extended family of proteoglycans: social residents of the pericellular zone. Curr Opin Cell Biol. 1989 Dec;1(6):1201–1218. doi: 10.1016/s0955-0674(89)80072-9. [DOI] [PubMed] [Google Scholar]
  9. Glass D. B., Smith S. B. Phosphorylation by cyclic GMP-dependent protein kinase of a synthetic peptide corresponding to the autophosphorylation site in the enzyme. J Biol Chem. 1983 Dec 25;258(24):14797–14803. [PubMed] [Google Scholar]
  10. Glass D. B., el-Maghrabi M. R., Pilkis S. J. Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases. J Biol Chem. 1986 Feb 25;261(6):2987–2993. [PubMed] [Google Scholar]
  11. Gould S. E., Upholt W. B., Kosher R. A. Syndecan 3: a member of the syndecan family of membrane-intercalated proteoglycans that is expressed in high amounts at the onset of chicken limb cartilage differentiation. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3271–3275. doi: 10.1073/pnas.89.8.3271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grobstein C., Cohen J. Collagenase: effect on the morphogenesis of embryonic salivary epithelium in vitro. Science. 1965 Oct 29;150(3696):626–628. doi: 10.1126/science.150.3696.626. [DOI] [PubMed] [Google Scholar]
  13. Inki P., Stenbäck F., Talve L., Jalkanen M. Immunohistochemical localization of syndecan in mouse skin tumors induced by UV irradiation. Loss of expression associated with malignant transformation. Am J Pathol. 1991 Dec;139(6):1333–1340. [PMC free article] [PubMed] [Google Scholar]
  14. Itano N., Oguri K., Nakanishi H., Okayama M. Membrane-intercalated proteoglycan of a stroma-inducing clone from Lewis lung carcinoma binds to fibronectin via its heparan sulfate chains. J Biochem. 1993 Dec;114(6):862–873. doi: 10.1093/oxfordjournals.jbchem.a124269. [DOI] [PubMed] [Google Scholar]
  15. Kjellén L., Lindahl U. Proteoglycans: structures and interactions. Annu Rev Biochem. 1991;60:443–475. doi: 10.1146/annurev.bi.60.070191.002303. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Lories V., Cassiman J. J., Van den Berghe H., David G. Multiple distinct membrane heparan sulfate proteoglycans in human lung fibroblasts. J Biol Chem. 1989 Apr 25;264(12):7009–7016. [PubMed] [Google Scholar]
  18. Marynen P., Zhang J., Cassiman J. J., Van den Berghe H., David G. Partial primary structure of the 48- and 90-kilodalton core proteins of cell surface-associated heparan sulfate proteoglycans of lung fibroblasts. Prediction of an integral membrane domain and evidence for multiple distinct core proteins at the cell surface of human lung fibroblasts. J Biol Chem. 1989 Apr 25;264(12):7017–7024. [PubMed] [Google Scholar]
  19. Nakanishi H., Oguri K., Takenaga K., Hosoda S., Okayama M. Differential fibrotic stromal responses of host tissue to low- and high-metastatic cloned Lewis lung carcinoma cells. Lab Invest. 1994 Mar;70(3):324–332. [PubMed] [Google Scholar]
  20. Nakanishi H., Oguri K., Yoshida K., Itano N., Takenaga K., Kazama T., Yoshida A., Okayama M. Structural differences between heparan sulphates of proteoglycan involved in the formation of basement membranes in vivo by Lewis-lung-carcinoma-derived cloned cells with different metastatic potentials. Biochem J. 1992 Nov 15;288(Pt 1):215–224. doi: 10.1042/bj2880215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nakanishi H., Takenaga K., Oguri K., Yoshida A., Okayama M. Morphological characteristics of tumours formed by Lewis lung carcinoma-derived cloned cell lines with different metastatic potentials: structural differences in their basement membranes formed in vivo. Virchows Arch A Pathol Anat Histopathol. 1992;420(2):163–170. doi: 10.1007/BF02358808. [DOI] [PubMed] [Google Scholar]
  22. Nakanishi Y., Ishii T. Epithelial shape change in mouse embryonic submandibular gland: modulation by extracellular matrix components. Bioessays. 1989 Dec;11(6):163–167. doi: 10.1002/bies.950110602. [DOI] [PubMed] [Google Scholar]
  23. Pierce A., Lyon M., Hampson I. N., Cowling G. J., Gallagher J. T. Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver. J Biol Chem. 1992 Feb 25;267(6):3894–3900. [PubMed] [Google Scholar]
  24. Rapraeger A., Jalkanen M., Bernfield M. Cell surface proteoglycan associates with the cytoskeleton at the basolateral cell surface of mouse mammary epithelial cells. J Cell Biol. 1986 Dec;103(6 Pt 2):2683–2696. doi: 10.1083/jcb.103.6.2683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Saunders S., Jalkanen M., O'Farrell S., Bernfield M. Molecular cloning of syndecan, an integral membrane proteoglycan. J Cell Biol. 1989 Apr;108(4):1547–1556. doi: 10.1083/jcb.108.4.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Spooner B. S., Thompson-Pletscher H. A., Stokes B., Bassett K. E. Extracellular matrix involvement in epithelial branching morphogenesis. Dev Biol (N Y 1985) 1986;3:225–260. doi: 10.1007/978-1-4684-5050-7_12. [DOI] [PubMed] [Google Scholar]
  27. Sugahara K., Ohi Y., Harada T., de Waard P., Vliegenthart J. F. Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. I. Six compounds containing 0 or 1 sulfate and/or phosphate residues. J Biol Chem. 1992 Mar 25;267(9):6027–6035. [PubMed] [Google Scholar]
  28. Woods A., Couchman J. R., Hök M. Heparan sulfate proteoglycans of rat embryo fibroblasts. A hydrophobic form may link cytoskeleton and matrix components. J Biol Chem. 1985 Sep 5;260(19):10872–10879. [PubMed] [Google Scholar]
  29. Woods A., Couchman J. R. Protein kinase C involvement in focal adhesion formation. J Cell Sci. 1992 Feb;101(Pt 2):277–290. doi: 10.1242/jcs.101.2.277. [DOI] [PubMed] [Google Scholar]
  30. Woods A., Couchman J. R. Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component. Mol Biol Cell. 1994 Feb;5(2):183–192. doi: 10.1091/mbc.5.2.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Woods A., McCarthy J. B., Furcht L. T., Couchman J. R. A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation. Mol Biol Cell. 1993 Jun;4(6):605–613. doi: 10.1091/mbc.4.6.605. [DOI] [PMC free article] [PubMed] [Google Scholar]

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