GLI1 is a key downstream transcription effector of the Hedgehog (Hh) signaling pathway that is involved in promoting cell growth, differentiation and tissue patterning in embryonic development. GLI1 over activation and its nuclear localization has also been linked to the increased aggressiveness of a number of cancers. It has previously been demonstrated that DYRKIA (dual-specificity tyrosine-regulated kinase 1A) can phosphorylate GLI1 and promote GLI1 nuclear localization and its transcriptional activity. Utilizing recombinant human GLI1 and DYRK1A proteins and phospho-peptide mass spectrometry, we demonstrated that GLI1 is phosphorylated by DYRKIA at Ser408, a phospho-site that falls within the putative nuclear localization sequence (NLS) of GLI1, suggesting a possible mechanistic role in modulating its translocation. Further, we showed that the Ser408 site on GLI1 was not phosphorylated in the presence of the selective DYRK1A inhibitor harmine. The data described herein provide the first identification of a DYRK1A-mediated site of phosphorylation on GLI1 within its NLS and may serve as a valuable mechanism for further understanding Hh signaling modulation. (C) 2017 Elsevier Inc. All rights reserved.