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, mon oclonal antibodies against proteins from the growth factor-mediated signall ing pathway were used to identify a set of 126-, 56-, 43-, and 30-kDa. prot eins phosphorylated on tyrosine at NO stimulation of murine fibroblasts ove rexpressing the human epidermal growth factor receptor. The band correspond ing to the 126-kDa protein was FAK. The 56-kDa protein was Src kinase, and the doubler 43- and 40-kDa protein corresponded to the extracellular-regula ted 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, sod ium nitroprusside, or with EGF did not change this association. We also det ected a basal constitutive association of Src kinase with FAK in HER14 cell s. 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 in crease in ERK1/ERK2 phosphorylation as assessed by a shift in its electroph oretic mobility and by increased phosphotyrosine immunoreactivity. Furtherm ore, NO-dependent activation of ERK1/ERK2 depended on the intracellular red ox status. Inhibition of glutathione synthesis was necessary to promote act ivation of the kinases. (C) 2000 Elsevier Science Inc.