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

Yasuyuki Kitaura, Shinji Matsumoto, Hirokazu Satoh, Kiyotaka Hitomi, Masatoshi Maki
2001 Journal of Biological Chemistry  
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
more » ... as 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. EXPERIMENTAL PROCEDURES Cell Culture-Jurkat cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (0.3 mg/ml), penicillin (100 units/ml) and streptomycin (100 g/ml) at 37°C
doi:10.1074/jbc.m008649200 pmid:11278427 fatcat:j62k3umt6rfdbaarweafplffsa