Ca2+-dependent translocation of the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells

The calcyclin-binding protein (CacyBP) binds calcyclin (S100A6) at physiological levels of [Ca2+] and is highly expressed in brain neurons. Subcellular localization of CacyBP was examined in neurons and neuroblastoma NB-2a cells at different [Ca2+](i). Immunostaining indicates that CacyBP is present in the cytoplasm of unstimulated cultured neurons in which resting [Ca2+](i) is known to be similar to50 nm. When [Ca2+]i was increased to above 300 nm by KCl treatment, the immunostaining was mainly apparent as a ring around the nucleus. Such perinuclear localization of CacyBP was observed in untreated neuroblastoma NB-2a cells in which [Ca2+]. is similar to120 nm. An additional increase in [Ca2+](i) to above 300 nm by thapsigargin treatment did not change CacyBP localization. However, when [Ca2+](i) in NB-2a cells dropped to 70 nm, because of BAPTA/AM treatment, perinuclear localization was diminished. Ca2+-induced translocation of CacyBP was confirmed by immunogold electron microscopy and by fluorescence of NB-2a cells transfected with an EGFP-CacyBP vector. Recombinant CacyBP can be phosphorylated by protein kinase C in vitro. In untreated neuroblastoma NB-2a cells, CacyBP is phosphorylated on a serine residue(s), but exists in the dephosphorylated form in BAPTA/AM-treated cells. Thus, phosphorylation of CacyBP occurs in the same [Ca2+](i) range that leads to its perinuclear translocation.