The Different Function of Single Phosphorylation Sites of Drosophila melanogaster Lamin Dm and Lamin C

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