title: Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis.
abstract: To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.
authors.affiliation: Laboratory of Glycobiology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. yamaoka@rinshoken.or.jp
authors.forename: Kazuko
authors.initials: K
authors.lastname: Yamaoka
authors.forename: Shinobu
authors.initials: S
authors.lastname: Imajoh-Ohmi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Fukuda
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Akita
authors.forename: Keiko
authors.initials: K
authors.lastname: Kurosawa
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Sanai
mesh_terms: D000818:Animals; D001358:Avian Sarcoma Viruses / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D019000:Phosphotyrosine / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
chemical_list: D010455:Peptides; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014357:Trypsin