The postmitotic growth suppressor necdin interacts with a calcium-binding protein (NEFA) in neuronal cytoplasm

Necdin, a growth suppressor expressed predominantly in postmitotic neurons, interacts with viral oncoproteins and cellular transcription factors E2F1 and p53. In search of other cellular targets of necdin, we screened cDNA li braries from neurally differentiated murine embryonal carcinoma P19 cells a nd adult rat brain by the yeast two-hybrid assay. We isolated cDNAs encodin g partial sequences of mouse NEFA and rat nucleobindin (CALNUC), which are Ca2+-binding proteins possessing similar domain structures. Necdin interact ed with NEFA via a domain encompassing two EF hand motifs, which had Ca2+ b inding activity as determined by Ca-45(2+) overlay. NEFA was widely distrib uted in mouse organs, whereas necdin was expressed predominantly in the bra in and skeletal muscle. In mouse brain in vivo, NEFA was localized in neuro nal perikarya and dendrites. By immunoelectron microscopy, NEFA was localiz ed to the cisternae of the endoplasmic reticulum and nuclear envelope in br ain neurons. NEFA-green fluorescent protein (GFP) fusion protein expressed in neuroblastoma N1E-115 cells was retained in the cytoplasm and partly sec reted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevati on of cytosolic Ca2+ levels. Thus, necdin and NEFA might be involved in Ca2 + homeostasis in neuronal cytoplasm.