Identification and characterization of a novel cytokine-inducible nuclear protein from human endothelial cells.
Vascular endothelial cells undergo profound changes upon cellular activation including expression of a spectrum of cell activation-associated genes. These changes play important roles in many physiological and pathological events. By differential screening of a cDNA library prepared from interleukin-1 alpha and tumor necrosis factor-alpha-stimulated human dermal microvascular endothelial cells, we have identified a novel cytokine-inducible gene, designated as C-193. The compiled cDNA sequence of C-193 is 1901 base pairs long and shows no significant homology with any known gene sequence. Genomic DNA analysis revealed that C-193 is encoded by a single gene, which is conserved in different mammalian species. The C-193 gene was localized to human chromosome 10 by Southern blot analysis of somatic cell hybrids. Multiple AT-rich mRNA decay elements were identified in the 3'-untranslated region. C-193 mRNA expression was rapidly and transiently induced by treatment with interleukin-1 alpha or tumor necrosis factor-alpha, reached a peak of expression about 16 h post tumor necrosis factor-alpha stimulation, and the induction of C-193 was protein synthesis independent. Lipopolysaccharide and cycloheximide were also potent inducers of C-193 mRNA. Therefore, C-193 represents a new addition to the primary response gene family. In vitro translation of C-193 yielded a 36-kDa protein product, consistent with the predicted open reading frame of 318 amino acids and a calculated molecular mass of 36 kDa for C-193 protein. The predicted protein sequence contains a basic amino acid cluster similar to a nuclear localization signal, four tandem repeats of ankyrin-like sequence, and multiple consensus protein phosphorylation sites. C-193 was engineered with a FLAG tag at its carboxyl terminus and transiently expressed in COS cells. Consistent with the presence of a putative nuclear localization signal, the C-193-FLAG protein was localized to the nucleus of transfected COS cells by indirect immunofluorescence microscopy. C-193-FLAG prepared in vitro was capable of binding DNA cellulose. These results indicate that C-193 protein may play an important role in endothelial cell activation.
Chu
W
W
Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Roche Research Center, Nutley, New Jersey 07110, USA.
Burns
D K
DK
Swerlick
R A
RA
Presky
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D002879:Chromosomes, Human, Pair 10; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009124:Muscle Proteins; D009687:Nuclear Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
C491304:ANKRD1 protein, human; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D009124:Muscle Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D003513:Cycloheximide