Signal transducer and activator of transcription 1 (STAT1) protein plays a pivotal role in various biological processes especially the regulation of innate and adaptive immune responses. Phosphorylation represents a key step in the activation of STAT1 and its transcriptional outcome. Binding of various extracellular ligands to their specific cell-surface receptors activates different phosphorylation of STAT1 followed by a distinct change of gene expression patterns. STAT1 is well-studied for its role in conducting the transcriptional response to interferons (IFNs), where it is activated by Janus kinase (JAK)–mediated phosphorylation of its Tyr⁷⁰¹ residue. However, the STAT1 function expands beyond its Tyr⁷⁰¹ phosphorylation. In this regard, we demonstrated that STAT1 serves as a proinflammatory effector downstream of toll-like receptor 4 (TLR4) endocytosis independently of IFN-ß signaling. In human macrophages, lipopolysaccharide (LPS)–bound TLR4 endocytosis activated noncanonical phosphorylation of STAT1 at Thr⁷⁴⁹, which altered its DNA target motif. Thr⁷⁴⁹–phosphorylated STAT1 promoted the expression of the gene encoding AT-rich interactive domain-containing protein 5A (ARID5A), which stabilizes interleukin 6 (IL6) mRNA. Moreover, Thr⁷⁴⁹– phosphorylated STAT1 directly enhanced the transcription of the gene encoding IL12B. By altering its DNA binding specificity, Thr⁷⁴⁹ phosphorylation confers STAT1 with proinflammatory properties in LPS-stimulated macrophages independent of its Tyr⁷⁰¹ phosphorylation. Thus, our study highlights the importance of understanding STAT1 phosphorylation status on its DNA binding specificity and transcriptional outcome, which may help in developing better therapeutic or diagnostic strategies targeting STAT1.

STAT1; Phosphorylation; Thr⁷⁴⁹, Tyr⁷⁰¹