Regulation of apoptosis by the p8/prothymosin α complex

p8 is a small-stress protein involved in several cellular functions including apoptosis. To identify its putative partners, we screened a HeLa cDNA library by using the two-hybrid technique and found that p8 binds the antiapoptotic protein prothymosin alpha (ProT alpha). Fluorescence spectroscopy, circular dichroism, and NMR spectroscopy showed that p8 and ProTa formed a complex. Binding resulted in important changes in the secondary and tertiary structures of the proteins. Because p8 and ProT alpha form a complex, they could act in concert to regulate the apoptotic cascade. We induced apoptosis in HeLa cells by staurosporine treatment and monitored the effects of knocking down p8 and/or ProT alpha or overexpressing p8 and/or ProTa on caspase 3/7 and 9 activities and on cell death. Transfecting ProT alpha or A small interfering RNAs increased the activities of both caspases and the number of apoptotic nuclei. However, transfecting both small interfering RNAs resulted in no further increase. Overexpressing p8 or ProT alpha did not alter caspase activities, whereas overexpressing both resulted in a significant reduction of caspase activities. These results strongly suggest that the antiapoptotic response of HeLa cells upon staurosporine treatment requires expression of both p8 and ProT alpha.