Puma(*)Mcl-1 interaction is not sufficient to prevent rapid degradation of Mcl-1.
Although Puma (p53 upregulated modulator of apoptosis) was known as a principal mediator of cell death in response to diverse apoptotic signals, the molecular mechanism underlying its proapoptotic regulation remains largely uncharacterized. Here we reported that myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family with a rapid turnover rate, interacts with Puma. The Puma/Mcl-1 interaction was verified by both yeast two-hybrid assay and co-immuno-precipation studies. Their binding sites were mapped to BH3 (Bcl-2 homology) domain of Puma and BH1 domain of Mcl-1, respectively. Mcl-1 and Puma was shown to colocalize at the mitochondria by immunostaining. The level of Mcl-1 was increased when coexpressed with Puma, indicating Puma is able to stabilize Mcl-1. Puma binding to Mcl-1 via its BH3 domain is the prerequisite for this effect, which is further supported by the finding that Puma mutant lacking BH3 domain no longer promotes Mcl-1 protein stability. This Puma-enhanced Mcl-1 stabilization was validated in vivo under non-overexpression conditions. We also showed that BH1 domain is essential for Mcl-1 to inhibit Puma-induced apoptosis, since Mcl-1 mutant lacking BH1 domain completely abrogates its protective function. In addition, we concluded that binding of Puma to BH1 domain of Mcl-1 is necessary, but not sufficient to prevent rapid degradation of Mcl-1. In addition to PEST (proline, glutamic acid, serine, and threonine) and BH1 domain, some additional degradation signal is expected to reside in the C-terminal region of Mcl-1. In conclusion, our results provide the first evidence that the interaction between Mcl-1 and Puma may represent a novel mechanism by which Mcl-1 prevents apoptosis by increasing its stability through binding to Puma.
Mei
Yide
Y
Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, People's Republic of China.
Du
Wenjing
W
Yang
Yonghui
Y
Wu
Mian
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009930:Organic Chemicals; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D012235:Rhodamines; D020798:Two-Hybrid System Techniques
D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D003574:Cytochrome c Group; D005456:Fluorescent Dyes; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D009930:Organic Chemicals; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012235:Rhodamines; C107472:red dye CMXRos; D049452:Green Fluorescent Proteins; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases