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Characterization of human SH2 domain‐containing protein 4A (SH2D4A) in HeLa cells
Author(s) -
Tamborlin Leticia,
Pereira Karina Danielle,
Meneguello Leticia,
Proença Andre Ricardo Gomes,
Luchessi Augusto Ducati
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb171
Subject(s) - gene , biology , gene isoform , open reading frame , microbiology and biotechnology , start codon , ww domain , complementary dna , promoter , genetics , gene expression , messenger rna , peptide sequence
The SH2 domain‐containing protein 4A (SH2D4A) has a single Src homology region 2 domain (SH2 domain) and a proline‐rich stretch on its C‐terminal. In humans, SH2D4A gene is located on chromosome 8p close to other genes associated with cancer. This region is deleted in patients with hepatocellular carcinoma (HCC) with poor outcomes, suggesting that depletion of SH2D4A could be involved with tumor progression. Besides, SH2D4A inhibits the STAT3 transcription factor activity mediated by interleukin 6 (IL‐6) signaling pathway, which interacts physically with STAT3, inhibiting the transcription of genes involved with cell proliferation, survival, and tumor development. Five transcripts corresponding to SH2D4A gene are described (variants 1, 2, 3, X1 and X2). The transcript variants 1 and 2 are predicted to encode the same isoform (isoform A), which has 454 amino acids and is considered canonical. The transcript variant 1 presents an upstream Open Reading Frame (uORF). Thus, this study aimed to characterize SH2D4A gene and protein in a model of tumor cells (HeLa cells), looking for new evidences to evaluate the involvement of this protein in development and tumor progression. First of all, we performed amplification of the cDNA by RT‐PCR and subsequent cloning. Thereby, it was possible to identify the transcript variants 1, 2 and X1 of SH2D4A, which was confirmed by sequencing of the samples. Analyzes of the sequenced samples revealed some mutations in SH2D4A gene, among them, the most significant was observed in the start codon, which results in the use of an alternative start codon to production of the protein. In addition, the presence of the uORF in SH2D4A variant 1 downregulates the protein production from transcript variant 1 in transfected cells. In fact, a depletion of the uORF increased the protein content compared to original variant 1. Our results suggest these mutations in SH2D4A gene and the presence of the uORF in transcript variant 1 may contribute to the abnormal characteristics observed in this type of cells. Support or Funding Information São Paulo Research Foundation (FAPESP)