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. 2017 Sep 23;491(3):767-772.
doi: 10.1016/j.bbrc.2017.07.107. Epub 2017 Jul 20.

Identification of a DYRK1A-mediated Phosphorylation Site Within the Nuclear Localization Sequence of the Hedgehog Transcription Factor GLI1

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Free PMC article

Identification of a DYRK1A-mediated Phosphorylation Site Within the Nuclear Localization Sequence of the Hedgehog Transcription Factor GLI1

Ben K Ehe et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

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 DYRK1A (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 DYRK1A 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.

Keywords: DYRK1A; GLI1; Hedgehog; Mass spectrometry; Nuclear localization; Phosphorylation.

Figures

Fig. 1
Fig. 1. Expression and purification of recombinant human GLI1 protein
FLAG-tagged GLI1 was expressed in HEK-293 cells and isolated using anti-FLAG-M2 affinity resin. (A) Western blotting with anti-GLI1 antibody. M = molecular weight markers; Elu = elution. (B) SDS-PAGE of purified FLAG-tagged GLI1.
Fig. 2
Fig. 2. In vitro DYRK1A kinase assays, gel slice isolation and phospho-mapping analysis of GLI1
Two (panels A and B) independent experiments were conducted. (A) Purified GLI1 was incubated in kinase buffer with (or without) DYRK1A and in the presence or absence of ATP. (B) Purified GLI1 was incubated in kinase buffer with (or without) DYRK1A, ATP, or with harmine. The protein bands corresponding to GLI1 (indicated by the black rectangles) were excised from the gels and treated with trypsin. Phospho-peptides were enriched and subjected to mass spectrometry for peptide identification and detection of phospho-sites. (C) Number of PSMs for APsISTVEPK phospho-peptide detected for each sample from A and B. ND = not done.
Fig. 3
Fig. 3. MS/MS fragmentation spectra for the unphosphorylated and phosphorylated APSISTVEPK GLI1 peptide
Mass spectra obtained without (A) and with (B) ATP addition during the in vitro kinase assay. The appearance of an additional peak with ATP is highlighted (red arrows). The mass shift corresponds to a difference of 79.9663 Da or 1 phosphorylation event.
Fig. 4
Fig. 4. Schematic of GLI1 domains and identified DYRK1A phosphorylation sites
The DYRK1A-mediated GLI1 phospho-peptide (residues 406-415) spanning the Ser408 site is highlighted in yellow and falls within the NLS. The two fragments of GLI1 (N-terminal (1-225) and C-terminal (394-1106)) shown previously by Mao et al. [15] to be phosphorylated by DYRK1A are shown bracketed (*). Dn – degron degradation signal; SU – SUFU-binding domain; ZF – Zinc finger DNA binding domain; NES – Nuclear export signal; NLS – Nuclear localization signal; TN - Transactivation domain. Residue numbering and domain designations based on Carpenter and Lo 2013 [20].

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