title: Tyrosine phosphorylation of NOS3 in a breast cancer cell line and Src-transformed cells.
abstract: We investigated the tyrosine phosphorylation of NOS3 by active Src. In a cell line derived from human breast cancer, BT474, we found activation of c-Src and tyrosine phosphorylation of NOS3. Phosphorylation of NOS3 was suppressed by treatment of BT474 with PP1, an Src kinase inhibitor, in a dose-dependent manner, suggesting that phosphorylation of NOS3 is catalyzed by active c-Src. Phosphorylation of NOS3 was further examined by a series of Src mutants. In cells expressing v-Src, substantial phosphorylation of NOS3 was observed, whereas NOS3 phosphorylation was not evident in cells expressing c-Src. Similarly, NOS1 was also phosphorylated in cells expressing v-Src. Consistently, in cells expressing a temperature-sensitive mutant of v-Src, NOS3 phosphorylation was temperature-dependent. Moreover, transforming mutant of c-Src, Y527Fc-Src, could activate NOS3 phosphorylation. In contrast, non-myristoylated form of v-Src, G2Av-Src and a kinase-inactive mutant of v-Src, K295Mv-Src, could not activate NOS3 phosphorylation. Taken together, our results suggest that active, membrane-bound form of Src can induce constitutive phosphorylation of NOS3.
authors.affiliation: Division of Cancer Biology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Takenouchi
authors.forename: Myat Lin
authors.initials: ML
authors.lastname: Oo
authors.forename: Takeshi
authors.initials: T
authors.lastname: Senga
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
authors.forename: Kou
authors.initials: K
authors.lastname: Miyazaki
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nimura
authors.forename: Michinari
authors.initials: M
authors.lastname: Hamaguchi
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D019556:COS Cells; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D009154:Mutation / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D014162:Transfection
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D011720:Pyrazoles; D011743:Pyrimidines; D019000:Phosphotyrosine; C496320:NOS1 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src)