Introduction
During the last two decades many proteins regulating neuronal death and survival have been described. The activity of these proteins is influenced by reversible phosphorylation. Thus, protein kinases and protein phosphatases are major regulators of cell viability. We have recently detected a protein histidine phosphatase (PHP) in eukaryotes 1 and have identified ATP citrate lyase (ACL) 2 and the β-subunit of heterotrimeric G-proteins as substrates of PHP. Since PHP is markedly expressed in brain tissue we attempted to unravel the physiological role of PHP in neuronal function.
Methods
PHP was expressed in E. coli. ACL was purified from rat brain and its activity determined by a 2-step enzyme assay. Localization of PHP and ACL was studied by western blot analysis and immunocytochemistry in brain, in primary cultures of neuronal cells (E18 and P1) and in neuronal and cholinergic cell lines. SH-SY5Y and SN56 cells were also used for overexpression (adenoviral vector) and knockdown (antisense and RNAi) of PHP. Cell viability was characterized by means of cell morphology and staining with Hoechst 33258.
Results
PHP was found in the cytosol of cells and co-localized with ACL. After in vitro phosphorylation of ACL at his-760 (autophosphorylation or phosphorylation by nucleoside diphosphate kinases), dephosphorylation of ACL by PHP reduced ACL enzyme activity. Addition of PHP-antisense to cells yielded a neuroprotective effect on staurosporine-induced cell death. On the other hand, the increase in PHP protein detected upon overexpression of PHP was in parallel to an increasing rate of damaged and dying cells.
Discussion
Our results provide evidence that PHP is involved in the regulation of cell viability. This might be attributed, at least in part, to its action on ACL, the enzyme producing acetyl-CoA thus providing the basis for lipid- and ACh synthesis. Cell viability depends on enzymatically active ACL, which is phosphorylated at his-760. It can be dephosphorylated, hence inactivated, by PHP. Accordingly, a PHP inhibitor might be a powerful neuroprotectant, particularly for cholinergic cells, because in these cells the inhibitor could improve lipid and acetylcholine synthesis.