Dephosphorylation-induced decrease of anti-apoptotic function of Bcl-2 in neuronally differentiated P19 cells following ischemic insults

It is known that Bcl-2 has a protective effect against neuronal ischemia. S ome reports speculate anti-apoptotic function of Bcl-2 depends not on the e xpression level but on the phosphorylation state. We found induction of apo ptosis and CPP32 activation by energy impairment (3-nitropropionic acid (3- NP)-treatment or glucose-deprivation) in the neuronally differentiated P19 cells. Time course study of cell viability following ischemic insults showe d that the number of viable cells decreased along with the increase in the amount of dephosphorylated Bcl-2 without obvious quantitative alteration of the protein. Then. we generated differentiated P19 cells overexpressing wi ld-type Bcl-2 (P19/wt.Bcl-2) or phosphorylation-negative Bcl-2 mutant (P19/ mut.Bcl-2), in which alanine was substituted for serine 70. When the cell v iability was examined within 23 h. P19/mut.Bcl-2 was mon vulnerable to ener gy impairment as compared with P19/wt.Bcl-2. In addition, overexpression of wild-type Bcl-2 inhibited DNA laddering and CPP32 activation induced by th e insults, while that of mutant Bcl-2 did not. These findings suggest that the phosphorylation state, as well as the expression level, of Bcl-2 plays an important role to modulate its protective effect against ischemic insult s. (C) 2000 Elsevier Science B.V. All rights reserved.