The different function of single phosphorylation sites of drosophila melanogaster lamin Dm and lamin C

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 (S 25E, S 45E, T 435E, S 595E). We also analyzed lamin C (A-type) and its mutant S 37E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R 64H 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 S 45E mutant was insoluble, in contrast to lamin C S 37E. Lamin Dm T 435E (C-terminal cdc2 site) and R 64H 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 S 45E and T 435E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T 435E was cytoplasmic and showed higher mobility in FRAP assay. © 2012 Zaremba-Czogalla et al.