The Function of Drosophila Lamins Phosphorylation Figure 1. Comparison of the conserved amino acid sequences located in the N-terminal and C-terminal fragment of lamins containing the cdc2 kinase site, using Clustal W. A schematic view of the lamin monomer with in vivo identified embryonic phosphorylation sites as well as NLS, Ig-fold and CaaX motif are shown. Identical amino acids are marked by black boxes, similar by shadowed boxes. Four conserved regions were identified in vertebrate lamins and three in Drosophila lamins. The first region in all lamins also contains the N-terminal cdc2 site (SPTR motif). The second region located at the very beginning of the tail domain is present in vertebrates only and contains their C-terminal cdc2 site (SPXXR motif). The Drosophila C-terminal cdc2 site is located partially in the third conservative region (T/SRAT/S sequences) – TPSR motif for lamin Dm and TPSGR motif in lamin C. There is also an alternative C-terminal cdc2 site in lamin C (S405 in SPGR motif ). LDm-D Drosophila melanogaster lamin Dm, LC-D lamin C from fruit fly, LA/C-H human lamin A/C, LB1-H human lamin B1, LB-M mouse lamin B, LB2-G chicken lamin B2, LB1-X Xenopus lamin B1, LB3-X Xenopus lamin B3, LA-X Xenopus lamin A. doi:10.1371/journal.pone.0032649.g001 sphorylation during apoptosis [29–30] and virus infection [31–33] has also been investigated. It was also suggested that phosphor- ylation could regulate lamin binding to chromatin [34]. In mouse myoblast cell line C2C12 lamin A is phosphorylated at S404 by Akt kinase in response to insulin [35]. Phosphorylation of human lamin A (S392) and B1 (S393) by cdk5 kinase can directly cause neuronal cell death observed in neurodegenerative diseases, including Alzheimer’s disease [36]. Unfortunately, so far almost nothing is known about the role of lamin phosphorylation in Drosophila melanogaster. Fruit fly has two lamin genes coding for lamin Dm and lamin C, which are equivalents of vertebrate B- and A-type lamins, respectively. There are at least three posttranslationally modified lamin Dm isoforms: Dm1, Dm2 and Dmmit [37–38]. Lamins Dm1 and Dm2 occur as an insoluble polymer that is mostly underlying the nuclear envelope during interphase [38]. Dm2 isoform arises from Dm1 by phosphorylation on the N-terminal site at or around S25 [38]. Both aforementioned isoforms, contrary to lamin C, interact with nucleic acids in vivo. During mitosis this interaction is lost [39]. Lamins Dm1 and Dm2 change during mitosis into a soluble form (lamin Dmmit) [37,39–40]. In vitro, lamin Dm polymers can be solubilized by cdc2, PKC (protein kinase C) and PKA kinase (protein kinase A) as well [11]. Early, syncytial embryo specific in vivo phosphorylation on soluble lamin Dm was demonstrated using mass spectrometry. The identified phosphopeptide suggested location of one of the phosphorylation sites on S45 residue within the sequence of typical, lamin N-terminal cdc2 kinase motif [41]. In older (6–18 h) embryos identified phosphopeptides suggested S19, S25, S595 and T435 (candidate cdc2/PKC/PKA site) as the late embryo in vivo phosphosites [41–42]. Our study on embryos and Kc cells demonstrated that in vivo more phosphorylation sites may be involved and that they may be distributed along the entire protein [37]. It is commonly known that one of the main functions of the phosphorylation of lamins is to facilitate nuclear envelope breakdown, which probably occurs due to effects on the ability of lamins to polymerize. But we do not know whether, and if so which, other functions and interactions of lamins, such as chromatin binding or lamin location during mitosis, are affected by mitotic phosphorylation. Moreover, we do not know anything about the role of the interphase phosphorylation of lamins and if there is any difference in the function of phosphorylation between B and A type lamins. Besides, for better understanding of lamin functions it is important to know what is the exact effect of phosphorylation of a particular site. We know that lamins may be phosphorylated on the same site by different kinases and typically up to three phosphorylation sites are modified at the same molecule (lamin Dm) so different combinations of phosphorylated sites may result in different phenotypic effects. It is essential to know the effect of single sites before we start to elucidate the function of simultaneous phosphorylation on many sites, as it is in vivo. That is why we started the project of generation of ‘‘stably phosphorylated’’ (pseudophosphorylated) lamin Dm mutants. The idea was to use a simple model system to mimic single phosphorylation of a particular known in vivo site by its conversion into glutamic acid (e.g. S25E, S45E, S595E, T435E in lamin Dm). Thus, we were able to see the effect of single site modification with respect to major lamin properties (e.g. polymerization, chromatin binding, location and transport). PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e32649