Abstract
Environmental stress (e.g. aniso-osmolarity and UV light), hypoxia/reoxygenation, and reactive oxygen species activate intracellular signaling cascades such as the "stress-responsive" mitogen-activated protein kinases and nuclear factor kappaB. We have recently shown that the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway is ligand-independently activated by hyperosmotic shock. In the present study, we show that besides STAT1 also the tyrosine phosphatase SHP2 became tyrosine-phosphorylated upon hyperosmolarity. SB 202190 and SB 203580 (specific inhibitors of p38) inhibited both STAT activation and tyrosine phosphorylation of SHP2 induced by hyperosmotic stress. Overexpression of wild-type p38 mitogen-activated protein kinase and its upstream activator mitogen-activated protein kinase kinase 6 (MKK6) resulted in an enhanced STAT1 tyrosine phosphorylation upon osmotic shock. Accordingly, overexpression of dominant negative mutants of p38 and MKK6 largely decreased hyperosmotic STAT1 activation and tyrosine phosphorylation of SHP2. Furthermore, we provide evidence that a genistein-sensitive tyrosine kinase different from Jak1 is involved in stress-activation of STAT1 and tyrosine phosphorylation of SHP2. These results strongly suggest that hyperosmotic shock activates STAT1 and SHP2 via p38 and its upstream activator MKK6.
Highlights
Environmental stress, hypoxia/reoxygenation, and reactive oxygen species activate intracellular signaling cascades such as the “stress-responsive” mitogen-activated protein kinases and nuclear factor B
We have recently shown that the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway is ligand-independently activated by hyperosmotic shock
Whereas extracellular signal-regulated kinase-type Mitogen-activated protein (MAP) kinases are preferentially activated by a variety of cell growth and differentiation stimuli and by hypoosmolarity, Jun N-terminal kinases (JNK) and p38 are primarily activated by various environmental stresses. p38 has substantial similarity to the S. cerevisiae HOG1 kinase, a yeast MAP kinase required for cellular osmoregulation (4)
Summary
Materials—Restriction enzymes were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Immunoprecipitation—Cells were washed twice with phosphatebuffered saline and solubilized in 1 ml of lysis buffer (0.5% Nonidet P-40, 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM EDTA, 20 mM glycerophosphate, 1 mM Na3VO4, 0.25 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin, and 15% glycerol) for 30 min at 4 °C. The Sepharose beads were washed three times with wash buffer (0.05% Nonidet P-40, 50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 1 mM NaF, 1 mM EDTA, 20 mM glycerophosphate, 1 mM Na3VO4, and 15% glycerol). P38 in Vitro Kinase Assay—Cells were lysed in a modified lysis buffer (0.5% Nonidet P-40, 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 20 mM -glycerophosphate, 1 mM NaF, 1 mM EDTA, 1 mM Na3VO4, 5 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin, 1 mM Pefabloc, and 15% glycerol), and immunoprecipitation was performed as described above using p38 rabbit polyclonal antibody (Upstate Biotechnology). The samples were resolved by 10% SDSPAGE and subjected to autoradiography
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