Taverna Elena, Francolini Maura, Jeromin Andreas, Hilfiker Sabine, Roder John, Rosa Patrizia
CNR, Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence on Neurodegenerative Diseases, Department of Medical Pharmacology, University of Milan, Via Vanvitelli 32, 20129 Milan, Italy.
J Cell Sci. 2002 Oct 15;115(Pt 20):3909-22. doi: 10.1242/jcs.00072.
Neuronal calcium sensor 1 (NCS-1) belongs to a family of EF-hand calcium-binding proteins and is mainly expressed in neurons and neuroendocrine cells, where it causes facilitation of neurotransmitter release through unknown mechanisms. The yeast homologue of NCS-1 has been demonstrated to interact with and regulate the activity of yeast phosphatidylinositol 4-OH kinase beta (PI4Kbeta). However, in neurons and neurosecretory cells NCS-1 has not unequivocally been shown to interact with PI4Kbeta. Here we have compared the subcellular distribution of NCS-1 and PI4Kbeta and investigated whether they are capable of forming complexes. In neurons, both proteins are widely distributed and are present in perikarya and, to a lesser extent, in nerve terminals. A consistent portion of NCS-1 and PIK4beta is cytosolic, whereas a portion of both proteins appears to be associated with the membranes of the endoplasmic reticulum and the Golgi complex. Very small amounts of NCS-1 and PI4Kbeta are present in synaptic vesicles. Our results further demonstrate that in neurosecretory cells, endogenous NCS-1 and PIK4beta interact to form a complex that can be immunoisolated from membrane as well as from cytosolic fractions. Moreover, both proteins can be recruited to membranes when cells are treated with nucleotide receptor agonists known to increase polyphosphoinositide turnover and concomitantly induce exocytosis of secretory vesicles. Finally, in PC12 cells overexpressing NCS-1, the amount of PI4Kbeta associated with the membranes is increased concomitantly with the increased levels of NCS-1 detected in the same membrane fractions. Together, these findings demonstrate that mammalian NCS-1 and PI4Kbeta interact under physiological conditions, which suggest a possible role for NCS-1 in the translocation of PI4Kbeta to target membranes.
神经元钙传感器1（NCS-1）属于EF手型钙结合蛋白家族，主要表达于神经元和神经内分泌细胞中，通过未知机制促进神经递质释放。已证明NCS-1的酵母同源物可与酵母磷脂酰肌醇4-羟基激酶β（PI4Kβ）相互作用并调节其活性。然而，在神经元和神经分泌细胞中，尚未明确显示NCS-1与PI4Kβ相互作用。在此，我们比较了NCS-1和PI4Kβ的亚细胞分布，并研究了它们是否能够形成复合物。在神经元中，这两种蛋白广泛分布，存在于胞体中，在神经末梢中的分布较少。NCS-1和PIK4β的一部分始终位于胞质中，而这两种蛋白的一部分似乎与内质网和高尔基体的膜相关联。突触小泡中存在极少量的NCS-1和PI4Kβ。我们的结果进一步证明，在神经分泌细胞中，内源性NCS-1和PIK4β相互作用形成复合物，该复合物可从膜以及胞质组分中免疫分离出来。此外，当用已知可增加多磷酸肌醇周转并同时诱导分泌小泡胞吐作用的核苷酸受体激动剂处理细胞时，这两种蛋白均可被募集到膜上。最后，在过表达NCS-1的PC12细胞中，与膜相关的PI4Kβ的量随着在相同膜组分中检测到的NCS-1水平的增加而增加。总之，这些发现表明哺乳动物的NCS-1和PI4Kβ在生理条件下相互作用，这表明NCS-1在PI4Kβ向靶膜的转运中可能发挥作用。
