Nitric oxide stimulates tyrosine phosphorylation of focal adhesion kinase, Src kinase, and mitogen-activated protein kinases in murine fibroblasts.
Nitric oxide (NO) can participate in cellular signaling. In this study, monoclonal antibodies against proteins from the growth factor-mediated signalling pathway were used to identify a set of 126-, 56-, 43-, and 40-kDa proteins phosphorylated on tyrosine at NO stimulation of murine fibroblasts overexpressing the human epidermal growth factor receptor. The band corresponding to the 126-kDa protein was FAK. The 56-kDa protein was Src kinase, and the doublet 43- and 40-kDa protein corresponded to the extracellular-regulated MAP kinases (ERK1/ERK2). The effects of NO on focal adhesion complexes were also investigated. FAK was constitutively associated with the adapter protein Grb2 in HER14 cells. Treatment of the cells with the NO donor, sodium nitroprusside, or with EGF did not change this association. We also detected a basal constitutive association of Src kinase with FAK in HER14 cells. In NO-treated cells, this association was stimulated. The doublet 43/40-kDa protein was identical to the ERK1/ERK2 MAP kinases. NO stimulated an increase in ERK1/ERK2 phosphorylation as assessed by a shift in its eletrophoretic mobility and by increased phosphotyrosine immunoreactivity. Furthermore, NO-dependent activation of ERK1/ERK2 depended on the intracellular redox status. Inhibition of glutathione synthesis was necessary to promote activation of the kinases.
Dept. of Biochemistry, Fundação Pró-Sangue Hemocentro de S. Paulo, Sao Paulo, Brazil. hpmonte@uol.com.br
H P
HP
Monteiro
J
J
Gruia-Gray
T M
TM
Peranovich
L C
LC
de Oliveira
A
A
Stern
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051380:GRB2 Adaptor Protein; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008970:Molecular Weight; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D015398:Signal Transduction; D014226:Triazenes / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D020030:Nitric Oxide Donors; D010750:Phosphoproteins; D011506:Proteins; C110881:S-nitro-N-acetylpenicillamine; D014226:Triazenes; C099612:NOC 9; D009599:Nitroprusside; D019000:Phosphotyrosine; D009569:Nitric Oxide; D019328:Buthionine Sulfoximine; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D010396:Penicillamine