Results Prediction of phosphorylation patterns on lamin Dm and lamin C Drosophila lamins may be used as a good model for studying the effect of phosphorylation on both types of lamins. Because to date there are no reports about experimentally confirmed phosphosites on the fruit fly lamin C, we used Clustal W for detection of potential, evolutionary conservative sites located in this protein. The results showed that S45 in lamin Dm and S37 in lamin C lie within the very conservative, classical cdc2 phosphorylation motif present in all lamins. The tail domain of Drosophila lamins contains two typical regions of phosphorylation sites: one located immediately next to the coiled-coil domain and near the NLS sequence and the second at the end of the tail domain. The site containing T435 represents a potential cdc2 site but this is not a typical, conservative cdc2 site present in vertebrate lamins (Figure 1). Instead, T435 lies near or within a less conserved region responsible for interaction of lamin Dm with histones H2A and H2B in vitro (TRAT sequence) [34] and the NLS sequence. Lamin C has no cdc2 motif in this region, and the nearest possible cdc2 site is represented by S405 (S405PGR). Thus Drosophila lamins lack a typical, conservative C-terminal cdc2 motif characteristic of vertebrate lamins but possess a classical N-terminal cdc2 site. Hence for the current study we decided to choose the following lamin proteins: wild-type lamin C and lamin C S37E mutant representing modification of the N-terminal cdc2 site as a control for A-type lamin. For lamin Dm study we used wild type protein, lamin Dm S25E, lamin Dm S45E, lamin Dm T435E and lamin Dm S595E. We also used lamin Dm R64H as an additional, control protein that is very well characterized in vitro, and unable to The Function of Drosophila Lamins Phosphorylation polymerize longitudinally [8–9,11,13,43]. All the above mentioned proteins were bacterially expressed and affinity purified to high homogeneity (Figure S1). Modification of single phosphorylation sites affects solubility of lamins In order to assess the secondary structure of bacterially expressed lamins, we analyzed their circular dichroism (CD) spectra (Figure S2). All wild type lamins and their mutants appeared to be well folded, containing domains with the helical coiled-coil conformation, and have the appropriate thermal denaturation curves. Lamin T435E was more resistant to thermal denaturation than wild type protein, while lamin Dm S595E was the least resistant. The calculated contents of secondary structures of analyzed proteins are shown in Table S1. The only well-documented function of specific in vivo phosphor- ylation of lamins is to affect lamin polymerization. We decided to confirm which particular lamin Dm phosphosites are responsible for modulation of polymerization using single pseudophosphoryla- tion mutants. We examined the solubility of lamins in a sedimentation assay using western blotting for lamin detection in the pellet fraction (insoluble, presumably polymerized or in paracrystals) and in the supernatant fraction (soluble, presumably unpolymerized form) (Figure 2). As an additional control we also used lamin Dm mutant R64H, which was reported as a polymerization deficient mutant [8–9,11,13,43]. Most lamin proteins demonstrated similar solubility. The exceptions were lamin Dm R64H, lamin Dm T435E and lamin C S37E. Lamin Dm mutant S45E representing the conserved N-terminal cdc2 phosphorylation site demonstrated polymerization similar to lamin Dm. Modification of the C-terminal cdc2 site on lamin Dm (T435) Figure 2. Phosphorylation of lamins changes their solubility in vitro. Panel A shows the typical result of sedimentation assay demonstrating polymerization ability of lamins. Proteins were allowed to polymerize for 60 min and centrifuged at 12 0006g for 20 min. Equal amounts of pellet and supernatant fractions were resolved onto 10% SDS PAGE followed by western blot with antibodies against D. melanogaster lamins. Panel B shows the diagram from the data obtained after quantification of the sedimentation assays. Data were obtained from three independent experiments. doi:10.1371/journal.pone.0032649.g002 PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e32649