Abstract
The function of site-specific phosphorylation of nucleophosmin (NPM), an essential Bax chaperone, in stress-induced cell death is unknown. We hypothesized that NPM threonine 95 (T95) phosphorylation both signals and promotes cell death. In resting cells, NPM exclusively resides in the nucleus and T95 is nonphosphorylated. In contrast, phosphorylated T95 NPM (pNPM T95) accumulates in the cytosol after metabolic stress, in multiple human cancer cell lines following γ-radiation, and in postischemic human kidney tissue. Based on the T95 phosphorylation consensus sequence, we hypothesized that glycogen synthase kinase-3β (GSK-3β) regulates cytosolic NPM translocation by phosphorylating T95 NPM. In a cell-free system, GSK-3β phosphorylated a synthetic NPM peptide containing T95. In vitro, bidirectional manipulation of GSK-3β activity substantially altered T95 phosphorylation, cytosolic NPM translocation, and cell survival during stress, mechanistically linking these lethal events. Furthermore, GSK-3β inhibition in vivo decreased cytosolic pNPM T95 accumulation in kidney tissue after experimental ischemia. In patients with acute kidney injury, both cytosolic NPM accumulation in proximal tubule cells and NPM-rich intratubular casts were detected in frozen renal biopsy tissue. These observations show, for the first time, that GSK-3β promotes cell death partly by phosphorylating NPM at T95, to promote cytosolic NPM accumulation. T95 NPM is also a rational therapeutic target to ameliorate ischemic renal cell injury and may be a universal injury marker in mammalian cells.