Peflin and ALG-2, members of the penta-EF-hand protein family, form a heterodimer that dissociates in a Ca2+-dependent manner.
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 (apoptosis-linked gene 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.
Mesh Terms:
Apoptosis Regulatory Proteins, Blotting, Western, Calcium, Calcium-Binding Proteins, Cell Membrane, Cell Nucleus, Chromatography, Gel, Cytoplasm, Cytoskeleton, DNA, Complementary, Dimerization, Humans, Jurkat Cells, Microscopy, Fluorescence, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Signal Transduction, Subcellular Fractions, Transfection
Apoptosis Regulatory Proteins, Blotting, Western, Calcium, Calcium-Binding Proteins, Cell Membrane, Cell Nucleus, Chromatography, Gel, Cytoplasm, Cytoskeleton, DNA, Complementary, Dimerization, Humans, Jurkat Cells, Microscopy, Fluorescence, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Signal Transduction, Subcellular Fractions, Transfection
J. Biol. Chem.
Date: Apr. 27, 2001
PubMed ID: 11278427
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