Data on peptides identified by mass spectrometry analysis of in vitro DYRK1A-mediated phosphorylation sites on GLI1

The data presented in this article support the accompanying research article “Identification of a DYRK1A-mediated phosphorylation site within the nuclear localization sequence of the hedgehog transcription factor GLI1” (Ehe et al., 2017) [1]. Although it has been demonstrated that DYRK1A (dual-specificity tyrosine-regulated kinase 1A) can phosphorylate the hedgehog pathway transcription factor GLI1 (GLIoma-associated oncogene homolog 1) and promote its nuclear localization, the DYRK1A-mediated sites of phosphorylation on GLI1 involved were not fully known. This article details the mass spectrometry methods and resulting dataset for the peptides identified from GLI1 when incubated with DYRK1A under varying conditions. The data include details of sequence coverage and all phospho-peptides identified.


Subject area
Biology More specific subject area Mass spectrometry analysis of phospho-peptide sites,

Value of the data
Use of high resolution mass spectrometry to identify phospho-peptides from GLI1 after DYRK1A incubation.
Identification of novel direct DYRK1A site of phosphorylation on GLI1.
Only data available for DYRK1A mediated phosphorylation of GLI1 within its NLS.

Data
The dataset presented here represents details of peptides that were identified by mass spectrometry from recombinant human GLI1 under varying conditions. We also present methodology for GLI1 expression and purification.
For GLI1 phospho-peptide analysis, two independent experiments were carried out. For study 1, the samples were GLI1 alone (sample 1), GLI1 þ DYRK1A þ ATP (sample 2) and GLI1 þ DYRK1A -ATP (sample 3) (Table 1). Samples were incubated for 30 min at 30°C, electrophoresed on SDS-PAGE, and the bands representing GLI1 were excised and the gel slices divided into two parts. For each sample, tryptic peptides were generated from both gel pieces for mass spectrometry analysis. The summary results and complete phospho-peptide analysis for the major site (Ser408) of DYRK1Amediated phosphorylation identified in Ehe et al. [1] are provided (Tables 1 and 2). Table 1 summarizes for study 1 the % sequence coverage, # peptides and # PSMs identified from GLI1 when treated under the differing conditions. For each run of each sample, approximately 50-70% coverage, 100 peptides, and 279-720 peptide spectral matches (PSMs) were identified. Observed peptide masses were compared to the GLI1 protein sequence (NCBI reference sequence: NM_005269.2) using MASCOT software [2]. Those GLI1 peptides identified with the highest number of PSMs (Z 4) are listed in Table 2. From these analyses, a number of high confidence phosphopeptides were identified with the majority present in all samples, suggesting those were related to basal GLI1 phosphorylation. The major difference between the different conditions was phosphorylation of the peptide APSISTVEPK at Serine 408 of GLI1. The unphosphorylated APSISTVEPK peptide was detected 10 to 13 times per run for each sample ( Table 3). The phosphorylated peptide was detected 6 times in each of the analyses of sample 2 (GLI1 þ DYRK1A þ ATP) and not in any other run (Table 3). For study 2, the same conditions as study 1 were replicated plus an additional sample; the samples were GLI1 alone (sample 1), GLI1 þ DYRK1A -ATP (sample 2) and GLI1 þ DYRK1A þ ATP (sample 3) and GLI1 þ DYRK1A þ ATP þ harmine (sample 4). The major site of DYRK1A-mediated phosphorylation on GLI1 was again identified as Ser408, with 10 of 11 peptides (MH þ [Da] ¼ 1108.52836790125) observed only in the þATP, þ DYRK1A sample (

GLI1 protein expression and purification
Recombinant human GLI1 protein was produced by transfection of the pCMV6 GLI1-myc-DDK plasmid (Origene, Rockville, MD) into HEK-293 cells (CRL-1573; ATCC, Manassas, VA). Cells were    For purification, cells were lysed in buffer supplemented with protease and phosphatase inhibitor cocktails. The cell extract was clarified by centrifugation at 14,000 rpm for 15 min. Anti-Flag affinity gel (100 µL) was transferred to a microcentrifuge tube and washed five times with TBS. The clarified cell extract was then added to the affinity gel and mixed by gentle inversion and incubated on a rocking platform for 2 h (4°C). After binding, the sample was centrifuged at 3000 rpm for 1 min to pellet the resin and the supernatant removed. The affinity gel was washed with 10 volumes of TBS-T (TBS with 0.05% Tween 20). Elution was carried out using 0.1 M acetate (pH 3.0), and the elution fractions were neutralized with 1 M Tris buffer (pH 9).

In vitro kinase assay and gel slice isolation
Purified GLI1 protein (2 µg) was incubated with recombinant human DYRK1A protein (Thermo-Fisher Scientific; 1 µg) in kinase buffer (25 mM Tris-HCl (pH 7.5) þ phosphatase inhibitors) with ATP (1 mM) for 30 min at 30°C. Controls included no DYRK1A, no ATP and plus 1 µM harmine (a selective DYRK1A inhibitor [3]). SDS-PAGE sample buffer was added to each sample, and following heating at 95°C for 5 min, samples were electrophoresed on a 4-12% Bis-Tris SDS-PAGE gel (Invitrogen; Carlsbad, CA) for 1 h at 120 V and bands detected with Coomassie blue staining. Gel bands were excised, reduced, alkylated, and digested with trypsin to generate tryptic peptides for mass spectrometry. Phospho-peptides were enriched using TiO 2 beads.

Mass spectrometry analysis
Mass spectrometry was performed at the UNC-CH Proteomics Center (Chapel Hill, NC, USA). TiO 2 beads were used to enrich for phosphopeptides. The tryptic peptides were then extracted, lyophilized, resuspended in 2% acetonitrile/98% (0.1% formic acid), and loaded onto a 2 cm long × 360 µm o.d. × 100 µm i.d. microcapillary fused silica pre-column packed with Magic 5 µm C18AQ resin (Bruker-Michrom Biosciences; Billerica, MA). After sample loading, the pre-column was washed with 95% solvent A (0.1% formic acid in water)/5% solvent B (0.1% formic acid in acetonitrile) for 20 min at a flow rate of 2 μL/min. The pre-column was then connected to a 360 µm o.d. × 75 µm i.d. analytical column packed with 22 cm of 5 µm C18 resin. The peptides were eluted (flow rate of 250 nL/min) by increasing the percentage of solvent B to 40% with a Nano-Acquity HPLC solvent delivery system (Waters Corp; Milford, MA). The LC system was directly connected through an electrospray ionization source interfaced to an LTQ Velos-Orbitrap ion trap mass spectrometer (Thermo Fisher Scientific; Waltham, MA), which determined the identity and phosphorylation status of the eluted peptides as previously described [4]. The mass spectrometer was controlled by Xcalibur software and operated in the data-dependent mode. Ions over the m/z range 400-2000 were recorded as the initial MS scans. The ten most abundant ions were automatically selected for collision-induced dissociation. All files were searched using MASCOT [2] (Matrix Science) using Proteome Discoverer against the protein of interest (GLI1, Origene ID RC201110). The search parameters included peptide mass tolerance of 10 ppm, fragment ion tolerance of 0.6 mass unit. The search allowed for variable oxidation of Met, phosphorylation of Ser, Thr, and Tyr, and carbamidomethylation of Cys. Each sample was run twice (n ¼ 2).