Protein phosphatase (PP) 2A (PP2A), a major
serine/threonine phosphatase highly active in the brain, is known to regulate programmed cell death by different mechanisms including downregulation of Ca++/
calmodulin-dependent
kinase IV (CaMK IV). Previous studies have shown that CaMK IV activity is increased following
cerebral hypoxia. In the present study, we tested the hypothesis that PP2A activity and expression in neuronal nuclei are decreased following
hypoxia in newborn piglets. PP and PP2A activities were determined in cerebral subcellular fractions spectrophotometrically using a
serine phosphopeptide in the presence or absence of microcystine. The activity of CaMK IV in neuronal nuclei was determined by 33P-incorporation into syntide 2 in the presence or absence of either 1 mM
EGTA or 0.8 mM CaCl2 and 1 mM
calmodulin. The expressions of PP2A and CaMK IV were measured using Western blot. Following
hypoxia, nuclear Ca++-dependent
kinase IV activity increased two-fold (P<0.001), whereas PP2A and PP activities significantly decreased (P<0.05) in the neuronal nuclei and membranes but not in the cytosol (P=NS). The distribution of the activity of PP2A was 60% in the cytosol, 35% in membranes and 5% in the neuronal nuclei. The expression of PP2A
protein showed a 14% increase and for CaMK IV
protein a 100% increase during
hypoxia. We propose that due to the decreased activity of PP and PP2A following
hypoxia in the neuronal nuclei there is a shift in the balance of the phosphorylation/dephosphorylation system toward increased phosphorylated state thereby increasing activity of the nuclear CaMK IV, modulator of programmed cell death. Since there is only slight increase in the PP2A
protein expression, we conclude that the changes observed in the activity of PP2A are due to
hypoxia-induced modification of the
enzyme itself. We also provide evidence that PP2A is a potential regulator of CaMK IV during
hypoxia.