These studies suggest that nucleoplasmic coilin, where the majority of the protein is found , may have a role in stress response pathways such as those caused by DNA damage. How 905586-69-8 phosphorylation of coilin impacts its putative role in these stress response pathways is unknown. In order to better clarify the role of phosphorylation on CB formation, we utilized coilin phosphomutants expressed both transiently and stably after induction in HeLa cells. We examined proliferation rates in these cells and monitored CB Abmole Company GANT61 formation both with normal and reduced levels of endogenous coilin. We have found that certain coilin phosphomutants inhibit cell proliferation while others have no effect, and this inhibition is associated with reduced CB number. Interestingly, two phosphomutants are degraded to an N-terminal fragment when expressed at levels close to that of the endogenous coilin, indicating a specific pathway for coilin degradation. These data demonstrate a crucial role for coilin phosphorylation in the formation of CBs. Previous results have demonstrated that coilin reduction inhibits cell proliferation . Since coilin is a phosphoprotein whose phosphorylation increases during mitosis , we would expect that any phosphomutant that alters CB formation or activity would negatively impact proliferation. To test for this possibility, we transiently transfected HeLa cells with various GFP-tagged coilin phosphomimic and phosphonull constructs in order to examine if any of the phosphomutants acted in a dominant negative manner over endogenous coilin. These constructs include a wild-type sequence as well as mutations changing 11 of the known phosphorylated residues to aspartic or glutamic acid or alanine . Additionally, three other constructs were used: T122 was mutated to glutamic acid , S489 was mutated to aspartic acid and S271/S272 were converted to aspartic acid . T122 and S271/272 were selected for mutation because MS/MS analysis have demonstrated that these residues are phosphorylated in both interphase and mitosis , suggesting an essential role for these modifications in coilin activity throughout the cell cycle. In contrast, S489 was selected for mutation because the phosphorylation of this residue appears to be enriched during mitosis when CBs are disassembled. We have previously shown that GFP-coilin properly localizes to CBs and the nucleoplasm and does not alter CB number when moderately expressed . In contrast, GFP-coilin ON and OFF expression alter normal coilin localization .