Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induce d apoptosis with unprecedented rapidity, more than 50% of primary hepatocyt es showing extensive surface budding and shrinkage of cytoplasm and nucleop lasm within 2 min. The apoptosis was retarded by the general caspase inhibi tor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodu larin into non-hepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells star ted to undergo budding typical of apoptosis within 0.5-3 min after injectio n. This was accompanied by cytoplasmic and nuclear shrinkage and externaliz ation of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosi s, Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 de ficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin, Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apopt osis was preceded by increased phosphorylation of several proteins, includi ng myosin light chain. The protein phosphorylation occurred even in the pre sence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase- inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.