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 libraries from neurally differentiated murine embryonal carcinoma P19 cells and adult rat brain by the yeast two-hybrid assay. We isolated cDNAs encoding partial sequences of mouse NEFA and rat nucleobindin (CALNUC), which are Ca(2+)-binding proteins possessing similar domain structures. Necdin interacted with NEFA via a domain encompassing two EF hand motifs, which had Ca(2+) binding activity as determined by (45)Ca(2+) overlay. NEFA was widely distributed in mouse organs, whereas necdin was expressed predominantly in the brain and skeletal muscle. In mouse brain in vivo, NEFA was localized in neuronal perikarya and dendrites. By immunoelectron microscopy, NEFA was localized to the cisternae of the endoplasmic reticulum and nuclear envelope in brain neurons. NEFA-green fluorescent protein (GFP) fusion protein expressed in neuroblastoma N1E-115 cells was retained in the cytoplasm and partly secreted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevation of cytosolic Ca(2+) levels. Thus, necdin and NEFA might be involved in Ca(2+) homeostasis in neuronal cytoplasm.
Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
N
N
Taniguchi
H
H
Taniura
M
M
Niinobe
C
C
Takayama
K
K
Tominaga-Yoshino
A
A
Ogura
K
K
Yoshikawa
D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D003593:Cytoplasm / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020832:EF Hand Motifs; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016350:Leucine Zippers; D051379:Mice; D016253:Microscopy, Immunoelectron; D008938:Mitosis; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009685:Nuclear Envelope / Q000737:chemistry; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000080866:Nucleobindins; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D006133:Growth Substances; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C000630652:Nucb1 protein, rat; D009687:Nuclear Proteins; D000080866:Nucleobindins; D011993:Recombinant Fusion Proteins; C069766:necdin; D002110:Caffeine; D002118:Calcium