Plasmids and cloning

For the generation of expression constructs encoding a N- or C-terminal twin-Strep and hemagglutinin (SH)-tag (pTO-SH) for affinity purification experiments or a N- or C-terminal FLAG-BirA*-tag51 for BioID experiments entry clones of a Gateway compatible clone collection (ORFeome v5.1) were used. The integration of the entry clones into the Gateway destination vectors was performed with an enzymatic LR clonase reaction (Invitrogen). Site-directed mutagenesis of Dyrk2 constructs was performed using Pfu Ultra High Fidelity DNA-polymerase according to the manufacturer’s instructions (Agilent Technologies). For the generation of the MultiBac vector expressing FLAG-tagged Dyrk2 (pFBDM-FLAG-Dyrk2) in SF9 cells, the coding sequence of Dyrk2 was amplified by PCR using oligonucleotides encoding the FLAG-tag and subcloned into pDNOR221 with BP clonase reaction (Invitrogen) (Supplementary Table 5). The DNA region encoding FLAG-Dyrk2 was inserted into pFBDM using NotI and HindIII restriction sites. The bacmid for the transfection of SF9 cells was generated by heat shock transformation of DH10Bac E. coli cells (#10361012, Invitrogen) with 2 µg pFBDM-FLAG-Dyrk2 and recovery at 37 °C for 8 h. Afterwards, cells were spread on agar plates containing Ampicillin, Kanamycin, Tetracycline, and Gentamycin. Grown colonies were used for the preparation of the bacmid DNA.

Tissue culture and DNA transfection

T-REx-HEK293 Flp–In (#R78007, Invitrogen) and T-REx-HeLa (#R71407, Invitrogen) cell lines were cultured in DMEM (4.5 g/l glucose, 2 mM l-glutamin) (Gibco) supplemented with 10% fetal bovine serum (BioConcept), 100 U/ml penicillin (Gibco), and 100 µg/ml streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO2. For DNA transfection, cells were treated with jetPrime (Polyplus) according to the manufacturer’s instructions.

Cell line generation

T-REx Flp–In cells were co-transfected with the respective expression plasmid and the pOG44 vector (Invitrogen) encoding the Flp-recombinase. Two days after the transfection, the selection of cells undergone recombination was initiated by addition of 15 µg/ml blasticidin C and 100 µg/ml hygromycin to the media for 2–3 weeks.

CRISPR/Cas9-mediated gene knock-out

CRISPR-guided RNAs (MM051/052 and MM063/064; Supplementary Table 5) were designed based on their specificity score retrieved from the Optimized CRISPR Design web tool (http://crispr.mit.edu) (gRNA 1 target sequence: 5′-ACCGGGGAGAAAACGTCAGT-3′, gRNA 2 target sequence: 5′-GGACAGCATTCATAGACGGC-3′). Annealed DNA oligonucleotides containing the target sequence were cloned into the hSpCas9 plasmid (pX458, Addgene) using BbsI restriction sites. T-REx-HeLa (#R71407, Invitrogen) cells were transfected with two hspCas9 constructs encoding gRNAs that target the third exon of the target gene DYRK2. The cell culture medium was replaced 4 h after transfection and cells were recovered for 72 h. For FACS sorting, 1 × 10e6 cells were gently detached from the tissue culture plate with 0.25% trypsin-EDTA (Gibco) and resuspended in PBS containing 1% FBS. GFP-positive cells were isolated by FACS (BD Facs Aria IIIu sorter) and single cells were sorted into each well of a 96-well plate. Cell clones were expanded for 3 weeks and then screened for deletion events by western blotting.

Western blot

Western blot analysis was performed with cells lysed in 100 µl lysis buffer (0.5% NP40, 50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 50 mM NaF supplemented with 1 mM PMSF and protease inhibitors (Sigma)). The cell lysate was cleared by centrifugation (15,000×g, 20 min) and boiled for 5 min after addition of 3× Laemmli sample buffer. The denatured sample was loaded on NuPAGE 4–12% Bis-Tris SDS-PAGE gels (Invitrogen) for gel electrophoresis and then transferred onto nitrocellulose membranes (Trans-Blot Turbo, BioRad). The following primary antibodies were used: anti-Dyrk2 (HPA027230, Sigma; 1:200), anti-DDB1 (D4C8, #6998, Cell Signaling; 1:1000), anti-Ubr5 (D608Z, #65344, Cell Signaling; 1:1000), anti-VprBP (#14966, D5K5V, Cell Signaling; 1:1000), anti-FLAG (F3165, Sigma; 1:5000), anti-HA (HA.11,901513, BioLegend; 1:5000), and anti-Actin (ab179467, Abcam; 1:1000). Proteins were detected by enhanced chemiluminescence (ECL, Amersham) using horseradish-peroxidase-coupled secondary antibodies (#7074, Cell Signaling (1:5000) and #115035003, Jackson ImmunoResearch (1:5000)). Uncropped images of the western blots are shown in the Source Data file.

Immunofluorescence

T-REx-HeLa cells (#R71407, Invitrogen) expressing GFP-tagged Dyrk2 variants were fixed on coverslips for 10 min with 600 μl 4 % Paraformaldehyde. After washing with PBS fixed cells were permeabilized with 0.2 % Triton-X 100 for 5 min, washed with PBS and then incubated with 600 μl 2% BSA for 30 min. Nuclear staining was performed with Hoechst (1:10,000, 10 mg/ml stock solution). Coverslips were mounted on the microscope slide with ProLong Gold antifade reagent (Invitrogen).

For the analysis of localization of Dyrk2 WT, KO, and SX mutant, more than 20 cells per condition were imaged on a wide-field Olympus MM microscope. Images were analyzed in Image J Fiji v1, cytoplasmic and nuclear signals were obtained by manual and automatic segmentation, respectively, and their ratio calculated.

Colony formation assay

T-REx-HeLa cells (#R71407, Invitrogen) were washed twice with PBS and then fixed with ice cold 100% methanol for 15 min. After fixation cells were washed with PBS and staining was performed with 5% Crystal violet (Sigma) solution for 20 min at room temperature. Then cells were washed with PBS until the dye was no longer draining out.

MTT assay for cell proliferation estimation

T-REx-HeLa cells (#R71407, Invitrogen) were seeded in 6-well plates and treated with 0.1 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma) for 3 h at 37 °C. The medium was removed and the converted dye was solubilized in 100% isopropanol. The absorbance of the converted dye was measured at a wavelength of 570 nm with background subtraction at 670 nm (Synergy HT, BioTek).

Cell cycle analysis

Cell cycle analysis was performed with the Propidium Iodide Flow Cytometry Kit (Abcam, ab139418) according to the manufacturer’s instructions. Briefly, T-REx-HeLa cells (#R71407, Invitrogen) were washed with PBS and then incubated with propidium iodide and RNase for 30 min at 37 °C. The stained cells were analyzed with the BD LSRFortessa (BD Biosciences) flow cytometer and data were analyzed with FlowJo v9.7.6.

Apoptosis assay

MDA-MB-231 cells (ATCC HTB-26) were analyzed for apoptosis using the Annexin V-FITC Apoptosis Staining/Detection Kit (Abcam, ab14085) according to the manufacturer’s instructions. Briefly, cells were harvested and resuspended in binding buffer. Then cells were incubated with Annexin V-FITC and propidium iodide for 5 min at room temperature followed by flow cytometry analysis with BD LSRFortessa (BD Biosciences) flow cytometer. The data were analyzed with FlowJo v9.7.6.

In vitro phosphorylation of NUP214

Commercial recombinant NUP214 (Aviva Systems Biology, OPCD05854) was incubated with 100 ng commercial recombinant Dyrk2 (Promega, V5090) in kinase reaction buffer (Promega, V9101) supplemented with ATP (0.1 μM) and DTT (1 mM) for 1 h at 37 °C. The reaction was stopped by adding 3× Laemmli buffer and heating up at 95 °C for 5 min.

In vitro ADP-Glo kinase assay

The in vitro kinase assay to estimate the activity of Dyrk2 mutants used in this study was performed with the ADP-Glo kinase assay kit (Promega, V9101) according to the manufacturer’s instructions. Briefly, equal amounts of recombinant Dyrk2 variants purified from SF9 insect cells were incubated in kinase reaction buffer (Promega, V9101) together with ATP (10 uM), DTT (50 μM) and 3 μg substrate (DYRKtide) (Promega, V9101) for 1 h at 37 °C. Then ADP-Glo reagent was added to stop the kinase reaction, and to convert remaining ADP to ATP and the sample was incubated for 40 min at room temperature. After that kinase detection, reagent was added followed by incubation for 60 min at room temperature. Luminescence was measured in white 96-well plates (ThermoFisher, 265302) with a plate reader (Synergy HT, BioTek) using 1 s integration time.

Affinity purification

The expression of SH-tagged bait proteins stably integrated in T-REx-HEK293 Flp–In cells was induced with 1 µg/ml doxycycline for 24 h. For affinity purification, four confluent 150 mm tissue culture plates were harvested and the cell pellet was snap-frozen. Then the cell pellet was lysed in 4 ml lysis buffer (0.5% NP40, 50 mM HEPES (pH 7.5), 150 mM NaCl, 50 mM NaF, 400 nM Na3VO4 supplemented with 1 mM PMSF, 1.2 µM Avidin, and protease inhibitor cocktail (P8849, Sigma)). The cleared cell lysate was incubated with Strep-Tactin beads (IBA LifeSciences) for 1 h on a rotation shaker. Upon washing two times with lysis buffer and three times with the same buffer but without protease inhibitor and detergent, the protein complexes were eluted from the Strep-Tactin beads with 2 mM biotin. Proteins of the eluate were precipitated with trichloroacetic acid and then dissolved in 8 M urea. Cysteine bonds were reduced with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) and alkylated with 10 mM iodoacetamide. The proteins were digested with 0.8 µg trypsin (V5112, Promega) over night followed by peptide clean-up with C18 UltraMicroSpin columns (The Nest Group). The dried peptides were dissolved in 2% acetonitrile and 0.1% formic acid.

BioID

One subconfluent (80%) 150 mm plate of T-REx-HEK293 Flp–In cells stably expressing FLAG-BirA*-tagged bait proteins was incubated for 24 h with 1 µg/ml tetracycline for protein expression. Then the media was replaced and the cells were incubated with 50 µM biotin for additional 24 h. After collection of the cells and centrifugation (400×g, 5 min), the cell pellet was snap-frozen. Lysis of the cell pellet was performed in 1 ml RIPA buffer (50 mM Tris–HCl (pH 8), 150 mM NaCl, 1% Triton-X 100, 1 mM EDTA, 0.1% SDS supplemented with 1 mM PMSF and protease inhibitor cocktail (Sigma)) followed by Benzonase (Sigma) treatment (250 U) at 10 °C for 30 min. The cleared lysate was then incubated with disuccinimidyl suberate (DSS) (Sigma) cross-linked Strep-Tactin beads (IBA LifeSciences) for 1 h on a rotation shaker. The beads were washed three times with RIPA buffer, three times with HNN buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 50 mM NaF), and two times with 100 mM NH4CO3. Proteins bound to the beads were denaturated with 8 M urea, reduced with 5 mM Tris(2-carboxyethyl)phosphine TCEP and alkylated with 10 mM iodoacetamide. The sample was diluted with 100 mM NH4CO3 to 4 M urea and proteins were digested on the beads with 0.5 µg LysC (Wako) for 3 h followed by dilution to 1 M urea and digestion by 0.8 µg trypsin over night. The digestion was stopped by addition of 5% formic acid and the peptides were purified by C18 UltraMicroSpin columns and dried in a speedvac. The dried peptides were dissolved in 2% acetonitrile and 0.1% formic acid.

Protein extraction and in-solution digest

For total proteome analysis T-REx-HeLa cells (#R71407, Invitrogen) were washed with ice cold PBS, scrapped off from the plate and snap-frozen in liquid nitrogen. The cell pellet was lysed in 8 M urea and sonicated three times for 1 min (Hielscher-Ultrasound Technology) followed by centrifugation at 18,000×g for 45 min to remove insoluble material. The protein amount of the cleared supernatant was measured by the Bicinchoninic acid (BCA) assay (Pierce) and 100 µg protein were subsequently reduced with 5 mM Tris(2-carboxyethyl)phosphine TCEP for 30 min at 37 °C and alkylated with 10 mM iodoacetamide for 30 min at 37 °C in the dark. The protein sample was diluted to 4 M urea with 100 mM NH4CO3 and digested by LysC (protease/protein ratio 1:100) for 4 h. The sample was further diluted to 1.5 M urea with 100 mM NH4CO3 and digested with Trypsin (protease/protein ratio 1:75) over night. The digestion was stopped by addition of 5% formic acid and peptides were purified by C18 columns (Sep-Pak, Waters). The desalted peptides were washed with 5% acetonitrile and 0.1% formic acid, eluted with 50% acetonitrile and 0.1% formic acid, and dried in a speedvac. The dried peptides were dissolved in 2% acetonitrile and 0.1% formic acid and iRT peptides (Biognosys) were added.

Phosphopeptide enrichment

T-REx-HeLa cells (#R71407, Invitrogen) were washed with ice cold PBS, scrapped off from the plate and snap-frozen in liquid nitrogen. The cell pellet was lysed in 8 M urea and sonicated three times for 1 min (Hielscher-Ultrasound Technology) followed by centrifugation at 18,000×g for 45 min to remove insoluble material. The protein amount of the cleared supernatant was measured by the Bicinchoninic acid (BCA) assay (Pierce) and 500 µg protein were then reduced with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) for 30 min at 37 °C and alkylated with 10 mM iodoacetamide for 30 min at 37 °C in the dark. The protein sample was diluted to 4 M urea with 100 mM NH4CO3 and digested by LysC (1:100) for 4 h. The sample was further diluted to 1.5 M urea with 100 mM NH4CO3 and digested with Trypsin (1:75) over night. The digestion was stopped by addition of 5% formic acid and the peptides were purified by C18 UltraMicroSpin columns. The dried peptides were dissolved in loading buffer for the enrichment of phosphopeptides (50% acetonitrile, 0.1% trifluoroacetic acid, 300 mg/ml lactic acid) and incubated with 5 mg TiO2 beads for 30 min at room temperature under strong shaking. The following steps were performed as previously described52. Phosphopeptides were eluted with 50 mM (NH4)2HPO4, pH 10.5 and the pH of the eluate was immediately adjusted to pH 2–3 with trifluoroacetic acid. Afterwards the sample was desalted with C18 columns (Sep-Pak, Waters) and dried in a speedvac. The dried phosphopeptides were dissolved in 2% acetonitrile and 0.1% formic acid and iRT peptides (Biognosys) were added.

Insect cell culture and transfection

SF9 cells (#11496015, Invitrogen) were cultured in SF9 insect cell media (Gibco) supplemented with PGS (Gibco) in an incubator (IKA shaker) at 27 °C and 300 rpm in the dark. After reaching a density of 2–3 × 10e6 cells/ml, the cells were diluted to 0.7 × 10e6 cells/ml. DNA transfection was carried out with GENEJuice transfection reagent (Sigma) according manufacturer’s instructions. Briefly, 2 × 10e6 cells were seeded in a 6-well plate and treated with 5 µg bacmid DNA added to the transfection reagent. After incubation for 72 h at 27 °C, the cell suspension was centrifuged for 5 min at 3000×g and the cleared supernatant containing the generated baculovirus was used for the infection of SF9 cells.

Protein expression and purification

For protein expression, 450 ml SF9 cell suspension culture at a density of 2 × 10e6 cells/ml were infected with baculovirus solution in a ratio 1:10 and incubated at 27 °C and 300 rpm in the dark for 3-4 days. At a cell viability of <70%, the SF9 cells were harvested by centrifugation for 5 min at 3000×g and the pellet was snap-frozen in liquid nitrogen. For protein purification, the cell pellet was lysed in 250 ml lysis buffer (50 mM HEPES, 150 mM NaCl, 50 mM NaF, 5% Glycerol supplemented with 400 nM Na3VO4, 1 mM PMSF, 1 mM Tris(2-carboxyethyl)phosphine (TCEP) and protease inhibitor cocktail (1:500) (Sigma)) and treated with 4000 U Benzonase (Sigma) for 30 min at room temperature. Afterwards, the lysate was sonicated and centrifuged to remove cell debris at 16,000×g for 20 min. The cleared lysate was incubated with FLAG M2 agarose beads (Sigma) for affinity purification of FLAG-tagged Dyrk2 over night on a rotation shaker at 4 °C. Afterwards, the beads were washed with lysis buffer without PMSF and protease inhibitors, and FLAG-tagged Dyrk2 was eluted with 1 mg/ml of FLAG peptide (ApexBio, Houston TX). The eluate was purified by size-exclusion chromatography with Superdex 75 10/300GL (GE Healthcare, Uppsala SWE) with running buffer (50 mM HEPES pH 8.0, 150 mM NaCl) to remove the excess of FLAG peptide. Samples were concentrated with 5 kDa molecular weight cut-off spin column (Vivaspin 500, Satorius) to a final protein concentration of 0.5 mg/ml determined by Bicinchoninic acid (BCA) assay. The quality of the purification (higher than 90%) was assessed by SDS-PAGE.

Cross-linking of Dyrk2 purified from SF9 insect cells

Purified Dyrk2 was cross-linked at a concentration of 0.5 mg/ml with 1 mM isotope labeled di-succinimidylsuberate (DSS-d0, DSS-d12) (CreativeMolecules Inc.) at 37 °C for 30 min as previously described28. The reaction was quenched with 100 mM NH4CO3 for 30 min. Afterwards, the sample was dried in a speedvac, re-dissolved in 8 M Urea, reduced with 5 mM Tris(2-carboxyethyl)phosphine (TCEP) and alkylated with 10 mM iodacetamide. For digestion, the sample was diluted to 1 M urea and trypsin (protease/protein ratio 1:50) was added over night. Digestion was stopped by acidification with 5% formic acid and peptides were purified by C18 clean-up. Dried peptides were dissolved in 20 μl 0.1% formic acid and 30% acetonitrile. Cross-linked peptides were enriched by peptide size-exclusion chromatography with Superdex Peptide PC 3.2/30 column (GE Healthcare, Uppsala) using running buffer containing 30% acetonitrile and 0.1% formic acid. SEC fractions were then dissolved in 5% acetonitrile and 0.1% formic acid, iRT peptides (Biognosys) were spiked to each sample before LC–MS/MS analysis for quality control and retention time alignment.

Cross-linking of Dyrk2 purified from mammalian cells

Eight subconfluent (80%) 150 mm plates of T-REx-HEK293 Flp–In cells stably expressing Strep/HA-tagged Dyrk2 were incubated for 24 h with 1 µg/ml tetracycline to induce protein expression. Cells were collected by centrifugation (400×g, 5 min) and snap-frozen. Then the cell pellet was lysed in 4 ml lysis buffer (0.5% NP40, 50 mM HEPES (pH 7.5), 150 mM NaCl, 50 mM NaF, 400 nM Na3VO4 supplemented with 1 mM PMSF, 1.2 µM Avidin, and protease inhibitor cocktail (P8849, Sigma)). The cleared cell lysate was incubated with disuccinimidyl suberate (DSS) (Sigma) cross-linked Strep-Tactin beads (IBA LifeSciences) for 1 h on a rotation shaker. Upon washing two times with lysis buffer and three times with the same buffer but without protease inhibitor and detergent, the cross-linking reaction of the affinity-purified Dyrk2 was performed by adding 1 mM isotope labeled di-succinimidylsuberate (DSS-d0, DSS-d12) (CreativeMolecules Inc.) at 37 °C for 30 min. The reaction was quenched with 100 mM NH4CO3 for 30 min and, after pelleting the beads, the supernatant was removed. Cross-linked Dyrk2 bound to the beads was denaturated with 8 M urea, reduced with 5 mM Tris(2-carboxyethyl)phosphine TCEP, and alkylated with 10 mM iodoacetamide. The sample was diluted with 100 mM NH4CO3 to 4 M urea and Dyrk2 was digested on the beads with 0.5 µg LysC (Wako) for 3 h, followed by dilution to 1 M urea and digestion by 0.8 µg trypsin over night. The digestion was stopped by addition of 5% formic acid and the peptides were purified by C18 UltraMicroSpin columns and dried in a speedvac. The dried peptides were dissolved in 2% acetonitrile and 0.1% formic acid.

Data acquisition for interaction analysis

LC–MS/MS analysis was performed on an Orbitrap Elite mass spectrometer (AP–MS) (ThermoScientific) coupled to an Easy-nLC 1000 system (ThermoScientific) and LTQ Orbitrap XL mass spectrometer (BioID–MS) (ThermoScientific) with Xcalibur software (4.1) (Thermo) coupled to an Easy-nLC II system (Proxeon). For samples derived from affinity purification peptides were separated on a Thermo PepMap RSLC column (15 cm length, 75 µm inner diameter) with a 60 min gradient from 5 to 35% acetonitrile at a flow rate of 300 nl/min whereas for BioID samples a gradient of 90 min (5–35% acetonitrile) was used. The mass spectrometer was operated in data-dependent acquisition (DDA) mode with the following parameters: one full FTMS scan (350–1600 m/z) at 120,000 resolution followed by MS/MS scans on the fifteen most abundant precursors with a charge state of +2 or higher, activation type = CID, isolation width = 1 m/z, normalized collision energy = 35%, activation Q = 0.25, activation time = 10 msec. The minimum signal threshold of precursors to induce MS/MS scans was set to 500 ion counts. For data acquisition, a dynamic exclusion for the selected ions was set: repeat count = 1, repeat duration 30 s, exclusion size list = 500, exclusion duration = 30 sec, exclusion mass width (relative to reference mass) = low 10, high = 10. For AP–MS, 10% and for BioID, 5% of the sample was injected.

Data acquisition for total proteome analysis

Samples for total proteome analysis were measured on a Sciex TripleTOF 6600 instrument (AB Sciex Instruments) with SCIEX Analyst v1.7 software equipped with a NanoLC Ultra 2D (Eksigent). Peptides were separated using a self packed C18 column (PicoTipTM Emitter, New Objective, Woburn, USA) (21 cm length, 75 µm inner diameter) with a 60 min gradient from 2 to 35% buffer B (98% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min. The mass spectrometer was operated in SWATH-mode using 64 variable windows between 400 and 1200 m/z (1 m/z overlap). The collision energy for each window was determined based on calculation for peptides with a charge state of 2+ adding a spread of 15 eV. For total proteome analysis, 1 µg peptides of the sample was injected.

Data acquisition for phosphoproteomic analysis

To generate a phosphopeptide-specific assay library, 26 phosphopeptide-enriched samples were acquired in high-resolution data-dependent acquisition mode on TripleTOF 6600 (AB Sciex Instruments). Phosphopeptides were separated by liquid chromatography (NanoLC Ultra 2D, Eksigent) at 0.3 ml/min flow rate interfaced to a NanoSpray III source (AB Sciex Instruments). As column material, a PicoTipTM Emitter (75 µm inner diameter) was in-house packed with C18 beads (MAGIC, 3 µm, 200 Å, Michrom BioResources, Auburn, USA) and cut to a length of 21 cm. Phosphopeptides were separated on a 120 min long linear gradient from 5% solvent A (2% acetonitrile and 0.1% formic acid) to 35% solvent B (98% acetonitrile and 0.1% formic acid). The twenty most intense precursor ions with a charge state between +2 and +5 were selected for CID fragmentation, and were excluded for re-fragmentation for 20 s. MS1 scan time was 300 ms over a mass to charge range of 360 to 1460, followed by 20 MS2 spectra measurements with 150 ms per spectra from 50 to 2000 m/z. For CID fragmentation, a collision energy spread of 15 eV was dynamically adjusted.

The same phosphopeptide-enriched samples were submitted to measurements in SWATH-mode on the TripleTOF 6600 instrument. For SWATH measurements, the gradient was shortened to 90 min from 5% solvent A (2% acetonitrile and 0.1% formic acid) to 35% solvent B (98% acetonitrile and 0,1% formic acid). Data were acquired in positive ion and high-sensitivity SWATH-mode, using 100 variable windows from 400 to 1250 m/z with 1 m/z overlap at the upper window boarder. A measurement time of 200 ms for MS1 precursor scans and 30 ms for each fragment ion scan was set, resulting in a 3.2 s duty time per cycle.

Data acquisition for cross-linking analysis

LC–MS/MS (DDA mode) was performed on Orbitrap Lumos Tribrid mass spectrometer (ThermoFischer) equipped with a Thermo easy-nLC1200 liquid chromatography system (ThermoFischer). Peptides were separated using reverse phase column (Acclaim PepMap RSLC C18 column, 2.0 µm, 75 µm*250 mm) across 60 min linear gradient from 7 to 35% (buffer A: 0.1% (v/v) formic acid, 2% (v/v) acetonitrile; buffer B: 0.1% (v/v) formic acid, 98% (v/v) acetonitrile). The data acquisition mode (data-dependent acquisition) was set to perform a cycle of 3 s with high-resolution MS scan in the Orbitrap (120,000 at 400 m/z) and MS/MS spectra in the ion trap. Charge state lower than 3 and bigger than 7 were rejected. The dynamic exclusion window was set to 25 s. Precursors with MS signal that exceeded a threshold of 5000 were allowed to be fragmented (CID, collision energy 35%). The ion accumulation time was set to 50 ms (MS) and 100 ms (MS/MS).

LC–MS/MS (target mode) was performed on Orbitrap Lumos Tribrid mass spectrometer (ThermoFischer) equipped with a Thermo easy-nLC1200 liquid chromatography system (ThermoFischer) using the same configuration as described for DDA mode. The data acquisition mode (PRM) was set to perform a MS1 scan followed by time scheduled targeted PRM scans acquired at variable resolution (60,000 and 120,000) fragmented as in the DDA acquisition. The quadrupole isolation window for the PRM events was set to 1.4 m/z units and the duration of the time scheduled windows were set to 2 min.

Data analysis on interactomics data

Acquired MS/MS scans were searched against the UniProtKB/Swiss-Prot protein database (10.05.2018) with the EulerPortal (ETH in-house software) workflow using the search engines X!TANDEM Jackhammer TPP (2013.06.15.1—LabKey, Insilicos, ISB), Comet (2016.01 rev. 3) and MyriMatch v2.1.138 considering a precursor mass tolerance of 15 ppm and a fragment mass error of 0.4 Da. Tryptic peptides with a maximum number of two missed cleavages were considered for the peptide identification search. Carbamidomethyl on cysteine residues as static modification was added.

High confident interactors of AP–MS experiments were determined by SAINTexpress53 with default parameters using spectral counts obtained from EulerPortal. Eighteen Strep/HA-GFP pulldowns processed and measured in the same way like the samples were used as controls for SAINTexpress scoring. Proteins with a SAINT score > 0.95 were filtered additionally against control runs of the CRAPome database (http://crapome.org/;54 411 pulldowns). IDs identified in > 15% of the CRAPome control runs and in the SH-GFP pulldowns of this study were removed to obtain the final set of high confident interactors.

High-confidence interactors of BioID–MS experiments were determined by SAINTexpress with default parameters as described. As control nine experiments with BirA*-GFP processed and measured in the same way like the samples were considered for the SAINT scoring. Furthermore, cells expressing the bait protein but not treated with biotin (nine experiments for Dyrk2, three experiments for Ubr5, DDB1, VprBP) were used as additional control for the SAINT scoring in order to identify and remove endogenously biotinylated proteins. IDs assigned with a SAINT score = 1 were considered as high confident interactors.

For the quantification of interactome changes in the AP–MS and BioID–MS network, LFQ MS1 intensities were determined by MaxQuant analysis v1.5.2.8 using default parameters and the UniProtKB/Swiss-Prot protein database25. Statistical analysis was performed by customized R scripts. Briefly, LFQ MS1 intensities were bait-normalized and missing values were imputed using random sampling from a distribution based on the 5th lowest quantile. In the following, fold changes and adjusted p-values were calculated.

Network visualization and GO analysis

The general layout of protein–protein interaction networks (Figs. 2a and 6a) was generated using Cytoscape (v3.6.0)55. To calculate the recall rate and represent already deposited interactions, we used as a reference database the Integrated Interaction Database (IID; release 2018.0518). Dot plots were generated using the ProHits-viz tool (https://prohits-viz.lunenfeld.ca/index.html56). GO analyses (Figs. 2b, 5g; Supplementary Fig. 2a) were carried out using DAVID (https://david.ncifcrf.gov/32) using primarily the BP(biological process)_DIRECT annotation in the Functional annotation tool and p-values for GO enrichment by a modified Fisher exact test. The network shown in Fig. 6a was prepared as follows: (i) first, we filtered proteins in our PPI and phosphoproteomics data such that they satisfy the following conditions: (A) They are present in the Cancer Census list (https://cancer.sanger.ac.uk/census, download 20190410); (B) They are regulated either at the phospho level (|log2FC| > 1|, peptide FDR < 0.05, and/or at the interactome level (|log2FC| > 1, adj. p-value < 0.05)). (ii) We mapped the known interactions among the selected proteins based on the integrated interactome database (IID v.2018-05; http://iid.ophid.utoronto.ca/). Of all interactions reported in IID, only those annotated as experimentally validated and with associated PubMed ID(s) were considered. In the graph, isolated nodes are those for which no interaction was retrieved. (iii) We performed a gene ontology analysis using DAVID (https://david.ncifcrf.gov/). Specifically, we considered the Biological Process domain (as opposed to Cellular Component or Molecular Function domains), and the default “Direct” category with default Homo sapiens background. To group GO terms, we used (i) shared parent terms identified with amiGO2 v.2.5.12 (which provides an inferred tree view of GO terms), (ii) and/or on shared assigned genes, (iii) and/or semantic similarity not captured in the amiGO2 inferred tree view.

Data analysis on total proteome data

DIA-SWATH data were analyzed with the OpenSWATH workflow57 implemented on the in-house EulerPortal platform after conversion of the raw SWATH wiff files into mzXML format by ProteoWizard (3.0.8851). Spectra were extracted using the combined human assay library with a fragment ion m/z extraction window of 0.05 Th and a retention time extraction window of 480 s. Detected features were aligned using TRIC with a target FDR of 0.01. If no peak group was detected, the area was re-quantified as described58. The OpenSWATH data output was further processed by the R package SWATH2stats59. The data were filtered with a global m-score cut-off of 3.9811E−5 resulting in a protein FDR of 0.0284 using an estimated fraction of false targets (FFT) of 0.46. The resulting data matrix was further filtered for proteotypic peptides. The following quantitative and statistical analysis was carried out by mapDIA v1.2.160. The filtered data matrix was normalized using total intensity normalization and an independent study design with a minimum correlation of 0.1 was chosen. For the calculation of fold changes and p-values, at least one peptide per protein and between 3–6 fragments per peptide were selected.

Generation of a phosphopeptide SWATH-assay library

The 26 in DDA mode measured files were used to generate a phosphopeptide-specific SWATH library. The raw MS files were converted to the mzXML file format with ProteoWizard (3.0.8851)61. For peptide identification, the data were searched using EulerPortal with the search engines X!TANDEM Jackhammer TPP (2013.06.15.1—LabKey, Insilicos, ISB), omssacl (version 2.1.9), and Comet (2016.01 rev. 3) against the human proteome (UniProtKB 10.05.2018) appended with reversed DECOY sequences for scoring. Tryptic peptides with a maximum number of two missed cleavages were considered for the peptide identification search. Carbamidomethyl on cysteine residues as static and phosphorylation on serine, threonine, and tyrosine, and oxidation on methionine residues as variable modifications were added. The maximum precursor mass error was 50 ppm and the fragment mass error was set to 0.1 Da. Identified peptide sequences were analyzed with the Trans-Proteomic Pipeline (TPP v4.7 POLAR VORTEX rev 0) applying PeptideProphet, iProphet, and Proteinprophet scoring. The search result was filtered at 0.01 FDR corresponding to an iprophet-peptide probability of 0.8829. To assess the localization of the phosphorylation site for each annotated spectra, the same strategy as reported in ref. 62 was used. In brief, a global false localization rate (FLR) was calculated using LuciPHOr230, allowing the classification of localized and non-localized phosphopeptides. All phosphopeptides with a lower FLR of 0.01 were annotated as localized, and all above the FLR threshold were annotated as non-localized phosphopeptides. For all non-localized phosphopeptides, the unique UniProtein identifier was expanded with “Phospho_1”. The spectral assay library was built using the Trans-Proteomic Pipeline as previously reported63 independently for localized and non-localized phosphopeptides and rejoined before DECOY and SWATH-assay generation. The final library contains 3239 localized and non-localized phosphopeptides, which origin from 1291 unique phosphoprotein groups.

Phospho-SWATH data quantification

The extraction of quantitative phosphopeptide data was conducted with the OpenSWATH workflow57 integrated in the EulerPortal tool and the Dyrk2-mutant-specific phospho-SWATH library generated for this project. The raw wiff files were converted to profile mzXML files with ProteoWizard (3.0.8851). All recorded phospho-SWATH maps were extracted with OpenSWATH applying an m/z fragment ion extraction window of 0.05 Th around the expected mass of the fragment ions and a retention time window of ±300 s after iRT-alignment. Scoring of the peaks was conducted with PyProphet applying 10 different scores. Detected features were mapped between runs, and re-aligned using a regression with a local minimum spanning tree model, with a 0.01 target FDR. If there was no feature identified within a run, the peptide and fragment signals were obtained by quantification of the respective background signal at the expected elution time of the peptide, allowing to boost the completeness of the resulting data matrix58. Next, the obtained intensities for all fragment features from OpenSWATH were processed with SWATH2stats59, allowing for elaborate filtering of the data matrix. Low quality features were removed with an m-score threshold of 0.01 and additionally a fragment feature had to be detected in at least 30% of all phospho-SWATH measurements. These conservative filtering resulted in a data matrix of 2887 quantified unique phosphopeptides from 1193 uniprot IDs across all conditions. Data were normalized using TIS (total ion spectra) normalization within the mapDIA framework60. Group comparison between each of the seven conditions against the wild-type Dyrk2 phospho signal was performed within mapDIA. This allowed us to obtain the log2FC values and to estimate the significance of change in intensity of phosphorylation of each given localized or non-localized phosphopeptide with a given adj. p-value (or FDR).

Analysis of cross-linking data

MS data were converted to mzXML format with msConvert and searched with xQuest/xProphet28 against a database containing the fasta sequence of Dyrk2 and its decoy sequence. Cross-linked peptides with a minimal length of 5 amino acids and a xQuest ld (linear discriminant) score higher than 25 were considered and selected for further analysis. The selected cross-linked peptides were quantified across different condition with a targeted proteomics approach (PRM = Parallel Reaction Monitoring). To perform the targeted analysis, we generated a library with the elution time of the precursor, m/z value and the charge of the cross-linked peptides (heavy and light form) identified by xQuest. Data analysis of acquired targeted cross-linked peptides were performed in Skyline v.4.164. The common transition (fragment ions that can be detected in the spectra of light and heavy cross-linked peptides) were imported as described65. Peptides were analyzed manually, and correct identification was assigned on the basis of the following criteria: (1) retention time matching with the identification by the xQuest analysis (±2 min), (2) co-elution of heavy and light cross-linked peptides, (3) matching of the peak shape and intensity for six common transitions of heavy and light cross-linked peptides. The abundance of cross-linked peptides was analyzed by summing the integrated area of twelve transitions per peptide (respectively six transition for the heavy and light form of the cross-linked peptide). Transitions with a signal to noise ratio <5 were filtered out for the quantification. Cross-linked peptides were normalized for the intensity of two non-cross-linked Dyrk2 peptides (acquired in PRM mode) and missing value were imputed with the minimum value identified in the analysis.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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