Research Article Phosphomimetic mutation of the mitotically phosphorylated serine 1880 compromises the interaction of the transmembrane nucleoporin gp210 with the nuclear pore complex Evgeny A. Onischenko a,b , Ellinor Crafoord a,b , Einar Hallberg a, ⁎ a Life Sciences, Södertörns University College, SE-141 89 Huddinge, Sweden b Department of Biosciences at Novum, Karolinska Institute, SE-141 86 Huddinge, Sweden ARTICLEINFORMATION ABSTRACT Article Chronology: Received 2 January 2007 Revised version received 12 April 2007 Accepted 14 May 2007 Available online 18 May 2007 The nuclear pore complexes (NPCs) reversibly disassemble and reassemble during mitosis. Disassembly of the NPC is accompanied by phosphorylation of many nucleoporins although the function of this is not clear. It was previously shown that in the transmembrane nucleoporin gp210 a single serine residue at position 1880 is specifically phosphorylated during mitosis. Using amino acid substitution combined with live cell imaging, time-lapse microscopy and FRAP, we investigated the role of serine 1880 in binding of gp210 to the NPC in vivo. An alanine substitution mutant (S1880A) was significantly more dynamic at the NPC compared to the wild-type protein, suggesting that serine 1880 is important for binding of gp210 to the NPC. Moreover a glutamate substitution (S1880E) closely mimicking phosphorylated serine specifically interfered with incorporation of gp210 into the NPC and compromised its post-mitotic recruitment to the nuclear envelope of daughter nuclei. Our findings are consistent with the idea that mitotic phosphorylation acts to dissociate gp210 from the structural elements of the NPC. © 2007 Elsevier Inc. All rights reserved. Keywords: Nuclear pore complex Nucleoporin Mitosis Phosphorylation FRAP Introduction The nuclear pore complexes (NPCs) are multiprotein channels located at the pore membrane, the fusion point between the outer and inner nuclear membranes [1]. The NPC conducts bi- directional signal-mediated transport of biological macromo- lecules between the nucleus and the cytoplasm. The frame- work of the NPC consists of multiple copies of approximately 30 different nucleoporins, the majority of which are conserved across eukaryotic species [2,3]. At the onset of mitosis in higher eukaryotes, the NPCs disassemble into distinct nucleoporin subcomplexes followed by dispersal of the nuclear membrane and solubilization of the nuclear lamina [4–6]. After chromosome segregation, the NPCs reassemble in the reforming nuclear envelopes (NEs) of daughter nuclei [1]. The nucleoporins are sequentially recruited to the reassembling NE starting from early anaphase until early G1 [7–9]. Many nucleoporins are specifically phosphorylated during mitosis [10–13]. Interestingly, in the lower eukaryote Aspergil- lus nidulans, in which the NPC undergoes only a partial disassembly, the mitotic release of Nup98 from the NPC is accompanied by phosphorylation and specifically depends on a kinase NIMA (acting downstream of the master mitotic EXPERIMENTAL CELL RESEARCH 313 (2007) 2744 – 2751 ⁎ Corresponding author. Fax: +46 8 608 4510. E-mail address: einar.hallberg@sh.se (E. Hallberg). 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.05.011 available at www.sciencedirect.com www.elsevier.com/locate/yexcr