Abstract
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.
Highlights
It is accepted that Ca2ϩ is one of the most versatile second messengers relaying information within cells to regulate their activities such as muscle contraction, secretory events, cell cycle, differentiation, gene expression, and apoptosis
This was confirmed by Western blotting of the immunoprecipitates of epitope-tagged peflin or apoptosislinked gene 2 (ALG-2) whose cDNA expression constructs were transfected to human embryonic kidney (HEK) 293 cells
The peflin protein was immunoprecipitated with monoclonal antibody (MoAb) P1G (Fig. 1B, lane 3) but not with an irrelevant MoAb 32C10A against human RECK (Fig. 1B, lane 2), which is not expressed in Jurkat cells
Summary
It is accepted that Ca2ϩ is one of the most versatile second messengers relaying information within cells to regulate their activities such as muscle contraction, secretory events, cell cycle, differentiation, gene expression, and apoptosis. 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.
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