title: Both ALG-2 and peflin, penta-EF-hand (PEF) proteins, are stabilized by dimerization through their fifth EF-hand regions.
abstract: ALG-2 (apoptosis-linked gene-2 protein) and peflin are Ca(2+)-binding proteins and belong to the penta-EF-hand (PEF) protein family, which includes calpain, sorcin, and grancalcin. ALG-2 forms either a homodimer or a heterodimer with peflin like other PEF proteins. In this study, we found that the fifth-EF-hand (EF-5) regions of both ALG-2 and peflin are essential for dimerization and their stabilities. Exogenously expressed EF-5-deletion (DeltaEF-5) mutants of ALG-2 and peflin were unstable and were not detected in HEK293 cells by Western blotting. In a pulse--chase experiment, the DeltaEF-5 mutants were rapidly degraded, but they were stabilized by treatment with a proteasome inhibitor, MG132. In MG132-treated cells, DeltaEF-5 mutants were recovered in the insoluble fractions. Transient coexpression of ALG-2 increased the peflin level. These results indicate that the absence of a fifth EF-hand results in rapid degradation by the proteasome. On the other hand, stable expression of exogenous peflin decreased the amount of endogenous peflin. The amount of peflin that can dimerize with ALG-2 seems to be restricted in mammalian cells.
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Kitaura
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Satoh
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takahashi
authors.forename: Hideki
authors.initials: H
authors.lastname: Shibata
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Maki
mesh_terms: D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D019281:Dimerization; D020832:EF Hand Motifs; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D017384:Sequence Deletion; D012995:Solubility; D020798:Two-Hybrid System Techniques
chemical_list: D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; C097753:PDCD6 protein, human; C121810:PEF1 protein, human; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde