Abstract
Osmotic shock induced transient stabilization of p53, possibly due to increased degradation of Mdm2. Stabilized p53 was activated by p38(MAPK), resulting in G(1) arrest through induction of p21(WAF1). Among the postulated phosphorylation sites involved in p53 stabilization or activation (Ser(15), Ser(20), Ser(33), and Ser(46)), only Ser(33) was phosphorylated. Furthermore, interaction of p53 with the transcriptional coactivator p300 was induced, and Lys(382) of p53 was acetylated. Although inhibition of p38(MAPK) did not prevent nuclear accumulation of p53, phosphorylation of Ser(33) was markedly suppressed by SB203580, a specific inhibitor of p38(MAPK). Under these conditions, acetylation of Lys(382) and induction of p21(WAF1) were also inhibited, and cells with elevated levels of p53 showed normal cell cycle progression. Activated p38(MAPK) phosphorylated endogenous p53 at Ser(33) in living cells. In stable transformants expressing dominant negative MKK6, an upstream protein kinase of p38(MAPK), p53 stabilization was induced normally following osmotic shock, but phosphorylation of Ser(33), acetylation of Lys(382), and induction of p21(WAF1) were almost completely inhibited. These results suggest that phosphorylation at Ser(33) by p38(MAPK) is critical for activation of p53 following osmotic shock. Phosphorylation of neither Ser(15) nor Ser(20) was needed in this activation.
Highlights
Cells respond to environmental stress with multiple defense systems for the maintenance of homeostasis or adaptation
We showed that p53 was activated in human cells by hyperosmotic treatment as determined by expression of p21WAF1, or cell cycle arrest
The results in this study indicate that the MKK6 3 p38MAPK (␣ or ) pathway is indispensable for osmotic shock-induced p53 activation
Summary
Cells and Hyperosmotic Treatment—Mori [40] is a primary cell strain of normal human fibroblasts. Cells were irradiated with x-rays and incubated for various periods at 37 °C These cells were prefixed with 3.7% formaldehyde for min at room temperature, washed with PBS, and fixed with 80% methanol for 10 min at Ϫ10 °C. Cells were stained with antibodies against p53 (PAb1801, Calbiochem) for 30 min at room temperature. Cells were cultured in plastic dishes (60 mm in diameter) for at least 2 days They were treated in hyperosmotic media for various periods. After a 30-min incubation with a fluorescein-conjugated anti-BrdUrd antibody (diluted 1:4; PharMingen), cells were treated with RNase A (100 g/ml) for 15 min at 37 °C and stained with propidium iodide (10 g/ml) for 30 min at room temperature. All points are displayed as the mean and standard deviation of triplicate assays
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