Serine 89 Phosphorylation Controls Nuclear Localization and Transcriptional Activity of ARID3B

Transcription factors that control stem cell programs are central drivers of cancer progression, metastasis, and therapy resistance. ARID3B, a DNA-binding protein overexpressed across multiple tumor types, expands the cancer stem cell population by regulating these pathways. Yet, how ARID3B is regulated remains largely unknown. Here, we uncover phosphorylation at Serine 89 as a critical switch controlling ARID3B localization and function. We used site-directed mutagenesis to generate phospho-dead (S89A) and phospho-mimetic (S89D) ARID3B constructs, and we generated a phospho-specific antibody for S89. With these tools, we showed that phosphorylation confines ARID3B to the nucleus in ovarian cancer and glioblastoma cells, as well as in human tissues, while unphosphorylated ARID3B can localize to the nucleus, cytoplasm, and membrane. Functionally, S89D mirrors wild-type ARID3B in regulating key transcriptional programs, whereas S89A diverges, consistent with altered subcellular localization. Chromatin immunoprecipitation confirms that direct gene regulation is enhanced in WT ARID3B and S89D compared to cells expressing S89A. Collectively, these findings reveal phosphorylation as a previously unrecognized molecular switch that dictates ARID3B’s localization and transcriptional activity, providing novel insights into cancer stem cell regulation and identifying a potential targetable vulnerability in aggressive tumors.