Akademik Veri Yönetim Sistemi
Turkish Journal of Medical Sciences, cilt.1, sa.1, ss.1-2, 2023 (SCI-Expanded)
Background/aim: T-cell acute lymphoblastic leukemia (T-ALL) is a type of leukemia characterized by the proliferation of immature T lymphocytes. NOTCH1 is one of the most frequently mutated genes in T-ALL. NOTCH1 expression in T-cell development depends on PHF6, which plays a tumor suppressor role in T-ALL. Several studies have shown that PHF6 expression is essential for NOTCH1 expression. Therefore, we investigate whether post-translational modification of PHF6 protein plays a role in the regulation of NOTCH1 expression and T-ALL cell line proliferation.
Materials and methods: We analyzed the amino acid sequence of PHF6 and found that a putative PKA phosphorylation motif RDRS199 was conserved in several vertebrate species and this site (S199) was expected to be phosphorylated according to PhosphoSite database. Therefore, we constructed a eukaryotic expression vector of human PHF6, and changed the codon 199 to the codon encoding the non-phosphorylatable Alanine and the phosphorylation-mimicking Aspartic acid via site-directed mutagenesis. After confirming ectopic expressions of the PHF6 vectors by western blot, we identified the effect of these proteins on NOTCH1 expression by western blot, leukemic cell proliferation by MTT assay and expressions of cell surface markers of T-cells by flow cytometry.
Results: Ectopic expression of wild-type PHF6 stimulated the formation of CD4+ T-cells. While expression of the wild-type PHF6 suppressed the growth of the leukemic cell line, this effect was diminished in both Alanine and Aspartic Acid mutants of PHF6. In addition, both mutants also seem to negatively affected NOTCH1 expression, although the effect of Alanine mutant was more severe.
Conclusion: Taken together, the different biological activities exerted by the conserved S199 phosphorylation-site mutants shown in this study implicate that signaling pathway(s) leading to differential phosphorylation of this residue may have a substantial effect on the activity of the PHF6 protein, thus may constitute a potential therapeutic target in T-ALL.