cyclic-gmp and Lupus-Erythematosus--Discoid

We used an autoimmune serum from a patient with discoid lupus erythematosus to clone a cDNA of 2808 base pairs. Its open reading frame of 2079 base pairs encodes a predicted polypeptide of 693 amino acids named CDA1 (cell division autoantigen-1). CDA1 has a predicted molecular mass of 79,430 Daltons and a pI of 4.26. The size of the cDNA is consistent with its estimated mRNA size. CDA1 comprises an N-terminal proline-rich domain, a central basic domain, and a C-terminal bipartite acidic domain. It has four putative nuclear localization signals and potential sites for phosphorylation by cAMP and cGMP-dependent kinases, protein kinase C, thymidine kinase, casein kinase II, and cyclin-dependent kinases (CDKs). CDA1 is phosphorylated in HeLa cells and by cyclin D1/CDK4, cyclin A/CDK2, and cyclin B/CDK1 in vitro. Its basic and acidic domains contain regions homologous to almost the entire human leukemia-associated SET protein. The same basic region is also homologous to nucleosome assembly proteins, testis TSPY protein, and an uncharacterized brain protein. CDA1 is present in the nuclear fraction of HeLa cells and localizes to the nucleus and nucleolus in HeLa cells transfected with CDA1 or its N terminus containing all four nuclear localization signals. Its acidic C terminus localizes mainly to the cytoplasm. CDA1 levels are low in serum-starved cells, increasing dramatically with serum stimulation. Expression of the CDA1 transgene, but not its N terminus, arrests HeLa cell growth, colony numbers, cell density, and bromodeoxyuridine uptake in a dose-dependent manner. The ability of CDA1 to arrest cell growth is abolished by mutation of the two CDK consensus phosphorylation sites. We propose that CDA1 is a negative regulator of cell growth and that its activity is regulated by its expression level and phosphorylation.

Topics: Amino Acid Sequence; Autoantigens; Base Sequence; Binding Sites; Blotting, Northern; Bromodeoxyuridine; Cell Division; Cell Membrane; Cell Nucleolus; Cell Nucleus; Chromosomal Proteins, Non-Histone; Cloning, Molecular; Cyclic AMP; Cyclic GMP; DNA; DNA-Binding Proteins; DNA, Complementary; Doxycycline; Fluorescent Antibody Technique, Indirect; HeLa Cells; Histone Chaperones; Humans; Immunoblotting; Lupus Erythematosus, Discoid; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nuclear Localization Signals; Phosphorylation; Precipitin Tests; Protein Kinase C; Protein Structure, Tertiary; Proteins; Sequence Homology, Amino Acid; Thymidine Kinase; Transcription Factors; Transfection; Transgenes