Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed in a stroma-inducing clone from a mouse Lewis lung carcinoma.
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.
Itano
N
N
Clinical Research Institute, National Nagoya Hospital, Japan.
Oguri
K
K
Nagayasu
Y
Y
Kusano
Y
Y
Nakanishi
H
H
David
G
G
Okayama
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018827:Carcinoma, Lewis Lung / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D007364:Intercalating Agents; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011509:Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053669:Syndecan-2; D014407:Tumor Cells, Cultured
D005353:Fibronectins; D007364:Intercalating Agents; D008081:Liposomes; D008562:Membrane Glycoproteins; D011509:Proteoglycans; C508246:Sdc2 protein, mouse; D053669:Syndecan-2