The S184 site contains a consensus cyclinE/Cdk2 kinase motif, but neither this site, nor the S202 residue, have been shown to be phosphorylated by phosphopeptide analyses. Regardless, the finding that mutation of these residues impacts coilin localization underscores the importance of the non-conserved middle region of human coilin. It is possible that these mutations facilitate the unmasking of a putative nucleolar localization signal located in the N-terminus of coilin . Tasocitinib JAK inhibitor Exposure of this NoLS may also account for the nucleolar accumulation of Nterminal fragments observed in the S489D and OFF stable cell lines . A clear understanding of how phosphorylation impacts coilin structure is lacking. Prediction programs, such as Disopred2 , indicate that coilin contains a large central region that is intrinsically disordered. In addition, the very C-terminus of coilin, which contains 6 phosphoresidues, is also predicted to be intrinsically disordered. In contrast, the N-terminal 96 aa, which contains the coilin selfassociation domain, and a region from aa 456�C563, which contains the coilin tudor domain , are predicted to have structure. Since most of the known phosphorylation sites in human coilin lie within regions of predicted intrinsic disorder, it may be that these modifications alter coilin interaction with other proteins as opposed to altering specific protein folds. In this study, we have tested different coilin phosphomutants for their ability to form CBs and affect 58880-19-6 cellular proliferation. These findings are summarized in Tables 1, 2, 3. The residues we have mutated have been shown by phosphopeptide analyses to be phosphorylated and are found in each of the groupings described above. Studies were conducted using both transiently transfected cells and stably transfected cell lines engineered to express physiological levels of different coilin phosphomutants upon induction. Transient transfection of GFPcoilin T122E, ON, S489D and S271/272D reduces cell proliferation , suggesting that these mutants act in a dominant negative manner over endogenous coilin when overexpressed. To reduce the possibility of overexpression artifacts as a consequence of transient transfection, we utilized the doxycycline- induced cell lines in combination with endogenous coilin reduction by RNAi.