Like many splicing factors, DAZAP1 is known to shuttle between the nucleus and cytoplasm 20,27,55,56, although it was mostly detected within the nucleus of cultured cells or spermatocytes 20,25,27 and in the cytoplasm of oocytes 17,24. We further examined if the localization of DAZAP1 is affected by its phosphorylation using immunofluoresecence in cells treated with MEK inhibitors. We find that in control cells treated with DMSO, DAZAP1 localizes in the nucleus as speckled structures in the majority of cells, whereas in some cells it can be found in both nuclear and cytoplasmic compartments. Consistent with our finding, DAZAP1 also colocalizes with hnRNP A1 and A2, which are known nucleo-cytoplasmic shuttling splicing factors that interact with DAZAP1. Co-localization was also confirmed with antibody against endogenous DAZAP1. However the DAZAP1 localization is distinct from the nuclear speckles containing SC35, an SR protein that promotes splicing. Upon treatment with MEK inhibitor, most of the DAZAP1 proteins were localized in perinuclear/cytoplasmic regions, while the hnRNPA1 is still found to be nuclear, suggesting that Erk-mediated protein translocation is specific to DAZAP1 rather than a general phenomenon for shuttling splicing factors. Serum starvation followed by the PMA treatment did not significantly alter the nuclear localization of DAZAP1 in HEK 293T cells.. To further confirm this result, we mutated the two threonine residues of DAZAP1 phosphorylated by Erk1/2 and examined localization of the mutant protein. While the majority of wild type and T2D DAZAP1 is in the nucleus, most T2A mutants are found in both the nucleus and cytoplasm, which is consistent with our observations using a MEK inhibitor. The inefficient localization of T2A mutant in the nucleus is also consistent with our finding that this mutation decreased the DAZAP1 splicing regulation activity. Taken together these results suggest that the Erkmediated phosphorylation probably controls the splicing regulatory activity of DAZAP1 via affecting its sub-cellular localization. In a recent study of mouse DAZAP1, Sasaki et al found that acetylation of Lys150 in the second PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19844160 RRM of DAZAP1 can affect its nucleocytoplasmic translocation 55. We found that such translocation my also be regulated by phosphorylation in the CTD of DAZAP1, suggesting the subcellular localization of DAZAP1 is controlled at multiple levels through different mechanisms. DAZAP1 controls cell proliferation Because the MEK/Erk pathway plays a key role in regulating cell JW 55 chemical information 
growth and malignant transformation, our observations suggest that DAZAP1-mediated splicing regulation might have an influence on cell growth. To test this, we generated stable cell lines with overexpressed DAZAP1 using a lung cancer cell line H157 and HEK 293T cells, and found that the wild type and phosphomimetic DAZAP1 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nat Commun. Author manuscript; available in PMC 2014 August 27. Choudhury et al. Page 10 inhibit cell growth in colony formation assays, while the T2A mutant had little effect. In addition, DAZAP1 expression also decreased anchorage independent growth of H157 cells as judged by soft agar colony assays. We also find that DAZAP1 can inhibit cell migration in a wound-healing assay, suggesting that DAZAP1-dependent splicing control may affect cell growth and migration. The decrease of cell growth in culture is commonly caused by increased apoptosis
