STAT3 phosphorylation at serine 727 activates specific genetic programs and promotes clear cell renal cell carcinoma (ccRCC) aggressiveness

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor mainly activated by phosphorylation in either tyrosine 705 (Y705) or serine 727 (S727) residues that regulates essential processes such as cell differentiation, apoptosis inhibition, or cell survival. we used microarrays to evaluate the effects of the STAT3 phosphomutants on global gene expression and identify the genes and pathways regulated by different STAT3 phosphorylation states in the 769-P cell line. we have generated human-derived ccRCC cell lines carrying STAT3 Y705 and S727 phosphomutants to identify genes and pathways regulated by pS727 that could be distinguished from those regulated by pY705 or by the combination of both. First, 769-P cells were depleted of endogenous STAT3 using shRNA and STAT3 WT form was then rescued. On this rescued STAT3 gene backbone, Y705 and S727 STAT3 phosphomutants were generated by introducing structurally similar amino acids that prevent (phosphoablative) or mimic (phosphomimetic) phosphorylation for each residue. A phosphomimetic substitution for Y705, however, was not possible since tyrosine is an aromatic amino acid and neither aspartic nor glutamic acid resembles the structure or charge density of a phosphotyrosine. To overcome this, we used interleukin-6 (IL6), a classic activator of the JAK/STAT3 pathway via pY705.