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.
Highlights
Lamins are the major components of the nuclear lamina, a dense, filamentous meshwork which provides structural support for the nuclear envelope (NE), a fraction of lamins are present in the nuclear interior as well
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
Lamin C has no cdc2 motif in this region, and the nearest possible cdc2 site is represented by S405 (S405PGR)
Summary
Lamins are the major components of the nuclear lamina, a dense, filamentous meshwork which provides structural support for the nuclear envelope (NE), a fraction of lamins are present in the nuclear interior as well. Lamins belong to the type V intermediate filaments. They contain a central a-helical rod domain flanked by a short Nterminal 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]. Lamins can 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 [8,9,10,11,12,13]
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