Puma(*)Mcl-1 interaction is not sufficient to prevent rapid degradation of Mcl-1

Oncogene. 2005 Nov 3;24(48):7224-37. doi: 10.1038/sj.onc.1208873.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Apoptosis Regulatory Proteins
  • BBC3 protein, human
  • Cytochrome c Group
  • Fluorescent Dyes
  • Myeloid Cell Leukemia Sequence 1 Protein
  • Neoplasm Proteins
  • Organic Chemicals
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Rhodamines
  • red dye CMXRos
  • Green Fluorescent Proteins
  • CASP9 protein, human
  • Caspase 9
  • Caspases