Abstract
The serine protease HtrA2/Omi is released from the mitochondria into the cytosol following apoptosis stimuli, leading to the programmed cell death in caspase-dependent and -independent manners. The function of HtrA2/Omi closely relates to its protease activity, which is required for cleavage of its substrate such as the members of the X-linked inhibitor of apoptotic protein family. However, the regulation of HtrA2/Omi by signaling molecule has not been documented. Here we report that serine/threonine kinases Akt1 and Akt2 phosphorylate mitochondria-released HtrA2/Omi on serine 212 in vivo and in vitro, which results in attenuation of its serine protease activity and pro-apoptotic function. Abolishing HtrA2/Omi phosphorylation by Akt through mutation of serine 212 to alanine (HtrA2/Omi-S212A) retains its serine protease activity and induces more apoptosis as compared with wild-type HtrA2/Omi. Conversely, HtrA2/Omi-S212D, a mutant mimicking phosphorylation, lost the protease activity and failed to induce the programmed cell death. Furthermore, the phosphorylated HtrA2/Omi fails to cleave X-linked inhibitor of apoptotic protein without interfering with their complex formation. In addition, Akt inhibits the release of HtrA2/Omi from the mitochondria into the cytoplasm in response to cisplatin treatment. These data reveal for the first time that HtrA2/Omi is directly regulated by Akt and provide a mechanism by which Akt induces cell survival at post-mitochondrial level.
Highlights
HtrA2/Omi-S212D, a mutant mimicking A role for an anti-apoptotic function of Akt at a post-mitophosphorylation, lost the protease activity and failed to induce chondrial level has been demonstrated [11,12,13]
Akt Inhibits the Apoptosis Induced by Mature HtrA2/Omi—The serine protease HtrA2/Omi is synthesized as a precursor that normally localizes at the mitochondria in healthy cell
The N-terminal amino acids preceding alanine 134 are cleaved and the resulted mature HtrA2/Omi is released from the mitochondria into the cytoplasm where it induces apoptosis (24 –29)
Summary
HtrA2/Omi pro-apoptotic function is regulated by Akt. HeLa cells were transfected with GFP-fused premature or mature HtrA2/Omi (Fig. 1A) together with or without wildtype and constitutively active Akt and treated with or without pan-caspase inhibitor z-VAD-FMK. The z-VAD-FMK only wild-type GST-HtrA2/Omi but not HtrA2/Omi-S212A (Fig. reduces the mature HtrA2/Omi-induced cell death by ϳ45% 3C), indicating Akt phosphorylation of HtrA2/Omi on serine (Fig. 1B).
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