Regulation of apoptosis by the p8/prothymosin alpha 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 (ProTalpha). Fluorescence spectroscopy, circular dichroism, and NMR spectroscopy showed that p8 and ProTalpha formed a complex. Binding resulted in important changes in the secondary and tertiary structures of the proteins. Because p8 and ProTalpha 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 ProTalpha or overexpressing p8 and/or ProTalpha on caspase 3/7 and 9 activities and on cell death. Transfecting ProTalpha or p8 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 ProTalpha 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 ProTalpha.
Institut National de la Santé et de la Recherche Médicale Unité 624, Stress Cellulaire, 163 Avenue de Luminy, Case 915, Parc Scientifique et Technologique de Luminy, 13288 Marseille Cedex 9, France.
Cédric
C
Malicet
Valentin
V
Giroux
Sophie
S
Vasseur
Jean Charles
JC
Dagorn
José Luis
JL
Neira
Juan L
JL
Iovanna
D017209:Apoptosis / Q000235:genetics; D051792:Basic Helix-Loop-Helix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D013057:Spectrum Analysis; D019311:Staurosporine / Q000494:pharmacology; D013947:Thymosin / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D051792:Basic Helix-Loop-Helix Proteins; D020169:Caspases; D009363:Neoplasm Proteins; D011498:Protein Precursors; D034741:RNA, Small Interfering; D019311:Staurosporine; D013947:Thymosin; C491643:NUPR1 protein, human; C040782:prothymosin alpha