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.
Mesh Terms:
Amino Acid Motifs, Amino Acid Sequence, Apoptosis, Apoptosis Regulatory Proteins, Binding Sites, Blotting, Western, Caspase 9, Caspases, Cell Line, Cytochrome c Group, Enzyme Activation, Fluorescent Antibody Technique, Indirect, Fluorescent Dyes, Gene Deletion, Green Fluorescent Proteins, Half-Life, HeLa Cells, Humans, Immunohistochemistry, Kinetics, Microscopy, Fluorescence, Mitochondria, Molecular Sequence Data, Mutagenesis, Site-Directed, Myeloid Cell Leukemia Sequence 1 Protein, Neoplasm Proteins, Organic Chemicals, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-bcl-2, Reproducibility of Results, Rhodamines, Two-Hybrid System Techniques
Amino Acid Motifs, Amino Acid Sequence, Apoptosis, Apoptosis Regulatory Proteins, Binding Sites, Blotting, Western, Caspase 9, Caspases, Cell Line, Cytochrome c Group, Enzyme Activation, Fluorescent Antibody Technique, Indirect, Fluorescent Dyes, Gene Deletion, Green Fluorescent Proteins, Half-Life, HeLa Cells, Humans, Immunohistochemistry, Kinetics, Microscopy, Fluorescence, Mitochondria, Molecular Sequence Data, Mutagenesis, Site-Directed, Myeloid Cell Leukemia Sequence 1 Protein, Neoplasm Proteins, Organic Chemicals, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-bcl-2, Reproducibility of Results, Rhodamines, Two-Hybrid System Techniques
Oncogene
Date: Nov. 03, 2005
PubMed ID: 16007132
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