Quantitative expression analysis of the cellular specificity of HECT-domain ubiquitin E3 ligases.
We evaluated the expression of 28 gene sequences with homology to the carboxy terminal of HECT E3 ubiquitin ligases in nine human cell lines using RT-PCR, to determine whether gene expression could be associated with cell-specific functions (HECT is "homologous to E6AP C-terminus"). In general, HECT-domain E3 ligases are constitutively expressed at low levels with a broad range between cell types. hecth3, 21, and 23 had higher levels in three leukocytic lines (Jurkat, MM6, THP1); hecth11 was more abundant in HepG2 and A495; and hecth15 and hecth12 were differentially expressed in lung fibroblasts derived from normal and severe emphysema patients (CCD16 and CCD29, respectively). Absolute quantitation showed that most HECT E3s have about 20-100 copies of mRNA per Jurkat cell. By comparison, UBCH7 (an ubiquitin-conjugating E2) is 10-fold more abundant in Jurkat cells and 30-fold more abundant than E2 UBCH5A. We interpret the broad range of transcript levels to be consistent with the hypothesis that the concentrations of E3 are important for ubiquitination selectivity, leading us to conclude that substrate activation is necessary but not sufficient for selectivity.
Scarafia
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Inflammatory Diseases Unit, Roche Bioscience, Palo Alto, California 94304, USA.
Winter
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Swinney
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D000595:Amino Acid Sequence; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D019169:Jurkat Cells; D008025:Ligases / Q000235:genetics*; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases
D012333:RNA, Messenger; D012313:RNA; D044767:Ubiquitin-Protein Ligases; D008025:Ligases