The Different Function of Single Phosphorylation Sites of Drosophila melanogaster Lamin Dm and Lamin C Magdalena Zaremba-Czogalla, Katarzyna Piekarowicz, Katarzyna Wachowicz, Katarzyna Kozioł, Magda Dubin´ ska-Magiera, Ryszard Rzepecki* Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland Abstract Lamins’ functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites (S25E, S45E, T435E, S595E). We also analyzed lamin C (A-type) and its mutant S37E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R64H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S45E mutant was insoluble, in contrast to lamin C S37E. Lamin Dm T435E (C-terminal cdc2 site) and R64H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N- terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S45E and T435E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T435E was cytoplasmic and showed higher mobility in FRAP assay. Citation: Zaremba-Czogalla M, Piekarowicz K, Wachowicz K, Kozioł K, Dubin´ ska-Magiera M, et al. (2012) The Different Function of Single Phosphorylation Sites of Drosophila melanogaster Lamin Dm and Lamin C. PLoS ONE 7(2): e32649. doi:10.1371/journal.pone.0032649 Editor: Michael Klymkowsky, University of Colorado, Boulder, United States of America Received October 27, 2011; Accepted January 28, 2012; Published February 29, 2012 Copyright: 2012 Zaremba-Czogalla et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by Polish Science Foundation (KBN) grant No. 3P04C 01725, and partially by Wroclaw Research Centre EIT+ under the project ‘‘Biotechnologies and advanced medical technologies’’ BioMed (POIG.01.01.02-02-003/08), financed by the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rzepecki@ibmb.uni.wroc.pl Introduction Lamins are the major components of the nuclear lamina, a dense, filamentous meshwork which provides structural support for the nuclear envelope (NE), although a fraction of lamins are present in the nuclear interior as well. Lamins serve as an organizing center for essential cellular processes such as transcrip- tion, DNA replication, cell differentiation, nuclear migration and others [1–4]. Mutations in nuclear lamina genes may cause a wide range of heritable human diseases generally termed laminopathies [5]. Lamins belong to the type V intermediate filaments. They contain a central a-helical rod domain flanked by a short N- terminal head domain and a carboxy-terminal tail domain, with NLS (nuclear localization signal or sequence) signal, conserved immunoglobulin fold and CaaX box (Figure 1) [6,7]. The central rod domain, which is necessary for the coiled-coil dimer formation, comprises four coiled-coil domains separated by flexible linker regions. Lamins can also associate longitudinally into head-to-tail polymers. In these interactions, binding sites located on the ends of the rod domain as well as in part of the head and tail domains near to the central domain are involved [8–13]. The level and pattern of phosphorylation of lamins vary throughout the cell cycle [14]. Phosphorylation of amino acid residues in the head and in the tail domain, located next to the central coiled-coil domain, inhibit lamin polymerization and might disrupt the nuclear lamina network during mitosis [11,15–20]. Mammalian mutant lamin A with removed cdc2 kinase (cell division control protein 2 homolog, also known as cyclin dependent kinase 1) phosphorylation sites caused aberrant mitosis when injected into mammalian cultured cells [21]. This was probably because of the lack of ability of lamins to depolymerize, but neither the mechanism nor any evidence has been revealed [17,20]. It is known that during mitosis the nuclear envelope structure is affected by mitotic microtubule-induced tearing of the nuclear lamina [22–23], although this must be accompanied by lamin phosphorylation on mitotic site(s) in order to weaken the strength of lamin polymers. Interestingly, it is possible that, for nuclear lamina depolymerization, not only proper (mitotic) sites for a particular lamin must be phosphorylated but also the interphase pattern of phosphorylation must be removed [24]. As well, at the end of mitosis, mitotic sites are dephosphorylated, and the nuclear envelope assembles again to encircle the decondensing chromatin. Some evidence has suggested that type 1 protein phosphatase (PP1) is the major mitotic lamin phosphatase responsible for removal of mitotic phosphates [25–26]. While the function of mitotic lamin phosphorylation is well characterized, very little is known about the possible role of specific lamin modification during interphase. One of them is the control of subcellular distribution of lamins by regulation of their uptake into the nucleus [27,28]. The role of lamin hyperpho- PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32649