Peflin and ALG-2, Members of the Penta-EF-Hand Protein Family, Form a Heterodimer That Dissociates in a Ca2+-dependent Manner

• Published in: The Journal of biological chemistry Vol. 276; no. 17; pp. 14053 - 14058
• Main Authors: Kitaura, Y, Matsumoto, S, Satoh, H, Hitomi, K, Maki, M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 27.04.2001
• Subjects: Apoptosis Regulatory Proteins; Blotting, Western; Calcium - metabolism; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - immunology; Cell Membrane - metabolism; Cell Nucleus - metabolism; Chromatography, Gel; Cytoplasm - metabolism; Cytoskeleton - metabolism; Dimerization; DNA, Complementary - metabolism; Humans; Jurkat Cells; Microscopy, Fluorescence; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Signal Transduction; Subcellular Fractions - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M008649200

Peflin, a newly identified 30-kDa Ca 2+ -binding protein, belongs to the penta-EF-hand (PEF) protein family, which includes the calpain small subunit, sorcin, grancalcin, and ALG-2 ( a poptosis- l inked g ene 2 ). We prepared a monoclonal antibody against human peflin. The antibody immunoprecipitated a 22-kDa protein as well as the 30-kDa protein from the lysate of Jurkat cells. Western blotting of the immunoprecipitates revealed that the 22-kDa protein corresponds to ALG-2. This was confirmed by Western blotting of the immunoprecipitates of epitope-tagged peflin or ALG-2 whose cDNA expression constructs were transfected to human embryonic kidney (HEK) 293 cells. Gel filtration of the cytosolic fraction of Jurkat cells revealed co-elution of peflin and ALG-2 in fractions eluting earlier than recombinant ALG-2, further supporting the notion of heterodimerization of the two PEF proteins. Surprisingly, peflin dissociated from ALG-2 in the presence of Ca 2+ . Peflin and ALG-2 co-localized in the cytoplasm, but ALG-2 was also detected in the nuclei as revealed by immunofluorescent staining and subcellular fractionation. Peflin was recovered in the cytosolic fraction in the absence of Ca 2+ but in the membrane/cytoskeletal fraction in the presence of Ca 2+ . These results suggest that peflin has features common to those of other PEF proteins (dimerization and translocation to membranes) and may modulate the function of ALG-2 in Ca 2+ signaling.