Mechanisms of site-specific dephosphorylation and kinase opposition imposed by PP2A regulatory subunits (2020)
Figure 6

Figure 6. PP2A-B56 is a regulator of ADAM17 mediated growth factor signaling and tumorigenesis.

A) EGFR activation in the ADAM17 variant expressing DLD-1 Adam17-/- cells upon H2O2 treatment, determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.

(B, C) B) Cell proliferation and C) Matrigel invasion of the ADAM17 variant expressing DLD-1 Adam17-/- cells. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.

D) Protein expression of the ADAM17 variants in the mouse breast cancer cell line 4T1 Adam17-/-, determined by Western blot. GAPDH was used as an internal loading control.

E) Average tumor volume ± SEM of 4T1 Adam17-/- cells expressing the ADAM17 variants injected into the mammary fat pad of Balb/c mice (n=10 for each group). Indicated significances are between the I762A and LEE tumors. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05. Mean and standard error of the mean indicated.

F) Survival curve of the tumor bearing mice injected with 4T1 Adam17-/- cells expressing the ADAM17 variants (n=10 for each group). The indicated significances are between the LEE and wt tumor bearing mice, and the LEE and I762A tumor bearing mice. Log-rank test was applied to test for significant differences. *p<0.05.

References

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Materials and Methods

Cell culture and reagents

Cancer cell lines DLD-1, HeLa and 4T1 (provided by the Barbara Ann Karmanos Cancer Institute) cells were maintained in DMEM GlutaMAX containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FCS (all from Thermo Fisher Scientific). Stable HeLa cell lines were generated using the T-Rex doxycycline inducible Flp-In system (Invitrogen) and cultivated like HeLa cells with the addition of 5 μg/mL Blasticidin and 100 μg/mL Hygromycin B. The DLD-1 and 4T1 cell line were regularly tested for mycoplasma infection and authenticated by STR profiling (Eurofins). E. coli DH5α and BL21(DE3) strains were maintained and propagated using standard microbiological procedures. The following drug concentrations were used: Thymidine 2.5 mM, Nocodazole 150 ng/µl, doxycycline 10 ng/ml unless otherwise stated.

CRISPR/Cas9 gene editing

DLD-1 Adam17 knockout cells were generated using the CRISPR-Cas9 system. Guide RNAs (gRNA) were designed using the WTSI genome editing tool (Hodgkins et al., 2015) and individually inserted in the vector pSpCas9(BB)-2A-GFP, as described (Ran et al., 2013). To determine the gRNA editing efficiency, cells were transfected with the pSpCas9(sgRNA)-2A-GFP vectors and verified by Indel Detection by Amplicon Analysis (IDAA) (Lonowski et al., 2017). Human gRNA 5 AAAGCGAGTACACTGTAAAA-3, targeting exon 4 and murine gRNA 5-ACAAAACTTG AGAGTCGTGG-3, targeting exon 3, showed the highest editing efficiencies and were subsequently transfected into DLD-1 and 4T1 cells respectively. GFP positive cells were single cell sorted, expanded and tested by qPCR and western blot for Adam17 knockout. Additionally, we screened positive clones for bi-allelic frameshifts using Sanger sequencing (Eurofins).

Expression constructs and cell line generation

Standard cloning techniques were used throughout. Point mutations were introduced by whole plasmid PCR. All constructs were fully sequenced. Synthetic DNA encoding the various B56 inhibitor sequences was purchased from GeneArt, Life Technologies. The pcDNA3.1 plasmid containing the murine cDNA of ADAM17 was provided by Prof. Dr. Stefan Rose-John (Kiel University, Kiel, Germany) and the I765A and M762L, D768E, P769E (LEE) mutants were generated by site directed mutagenesis. ADAM17 wt, I765A and LEE inserts were cloned into the sleeping beauty transposon vector v359 (Kowarz et al., 2015)

and transfected into the DLD-1 A17-/- or 4T1 A17-/- cells using Fugene HD (Promega), and selected with 2 µg/ml Puromycin (Sigma) for 6 days. For induction of ADAM17 expression, 10 ng/ml Doxycycline (Sigma) was added to the cell culture medium and incubated for 24 h. Detailed mutagenesis and cloning strategies are available upon request.

Antibodies

The following antibodies were used at the indicated dilutions: c-Myc (9E10, sc-40, 1:1000, Santa Cruz Biotechnology), Separase (A302-215A, 1:2000, Bethyl Laboratories), KIF4A (A301-074A, 1:1000, Bethyl Laboratories), Axin1 (#2087, 1:1000, Cell Signaling Technology), B56α (610615, 1:3000, BD Biosciences), BubR1 (A300-995A,1:1000, Bethyl Laboratories), PP2A catalytic subunit (05-421,1:2000, Millipore), PP2A scaffold subunit (#2041, 1:1000, Cell Signaling Technology) GFP (ab290, 1:4000, abcam), FoxO3 pS413 (#8174, 1:1000, Cell Signaling Technology), FoxO3 rabbit polyclonal α-pS253 (Raised against peptide CAPRRRAV(pS)MDNS; 1:500, Moravian Biotechnology), Anti-RFP (1:1000; MBL, FM005), Anti-GFP (1:1000; Roche, 11814460001), Rabbit anti-ADAM17 (1:1000, abcam, 2051), Rabbit anti-ADAM17 (1:1000, abcam, 39162), Rabbit anti-EGFR (1:1000, cell signaling, 2232), Rabbit anti-pEGFR Y1068 (1:1000, cell signaling, 2234), Mouse anti-Transferrin receptor (1:1000, Invitrogen, 136800), Mouse anti-GAPDH (1:5000, Sigma, G8795), rabbit anti-GFP (1:3000, Takara Bio Clontech, 632592), Donkey anti-rabbit-HRP (1:2000, GE-Healthcare, NA934), Sheep anti-mouse-HRP (1:2000, GE-Healthcare, NXA931), H3pS10 (06-570, 1:1000, Millipore).

Protein expression and purification

Cdc20 and ADAM17 fusion proteins, were cloned into pGEX-4T-1 to generate N-terminally GST-tagged fusion proteins. Constructs were transformed into BL21 (DE3) cells and expression was induced by addition of 0.5 mM IPTG at 18 degrees overnight. Following resuspension in buffer L (50 mM Tris pH 7.5; 300 mM NaCl; 10% glycerol; 0.5 mM TCEP, 1X complete EDTA-free tablets (Roche)) the sample was lysed using a high pressure homogenizer (Avestin). Lysate was clarified by centrifugation and loaded on a GSTrap HP 5 ml column and washed with buffer L to baseline absorbance at 280 nm. The proteins were eluted with buffer L containing 20 mM glutathione using a 20 CV gradient and the peak fractions pooled and collected. The peak fractions were concentrated on a vivaspin 20 and loaded on a Superdex 75 16/60 GL column equilibrated with buffer GF (50mM NaP pH=7.5; 150 mM NaCl; 10% glycerol; 0.5mM TCEP). Relevant fractions were pooled and flash frozen and stored at -80.

The B56 inhibitors and ctrl inhibitor was cloned into the pET30 expression vector and expressed in E. coli BL21 (DE3) cells as described above. Cells were suspended in buffer U (100 mM Tris pH=8.0; 300 mM NaCl; 7.4 M Urea) and sonicated followed by centrifugation for 2x25 min and sample filtered. The urea concentration was lowered to 1M by sequential dialysis (6M-3M-1M Urea) followed by centrifugation. The lysate was filtered and applied to a 5ml Ni-NTA affinity column and bound proteins eluted with an immidazole gradient. Peak fractions as measured by UV spectroscopy were pooled and the NaCl concentration lowered to 50 mM NaCl and applied to a MonoQ column. A gradient from 50 mM to 1000 mM NaCl was applied and peak fractions collected and pooled. Subsequently the pool was run on a superdex 75 16/60 column equilibrated with (50 mM Tris pH=7.5, 100 mM NaCl) and peak fractions pooled. Recombinant B56α, GST-Arpp19 and MASTL was produced as described previously (Hertz et al 2016; Hein et al 2017).

Isothermal titration calorimetry (ITC)

Recombinant B56α and B56 inhibitors were extensively dialyzed against ITC buffer 50 mM sodium phosphate pH 7.5, 200 mM NaCl, 0.5 mM TCEP. All experiments were performed on an Auto-iTC200 (Malvern Panalytical) instrument at 25 °C. The B56α inhibitors were loaded into the syringe and titrated into the calorimetric cell containing B56α (direct titrations) or vice versa (reverse titrations). Control experiments with either the inhibitors or B56α injected in the sample cell filled with buffer were carried out under the same experimental conditions. These control experiments showed negligible heats of dilution in all cases. The titration sequence consisted of a single 0.4 μl injection followed by 19 injections, 2 μl each, with 150 s spacing between injections to ensure that the thermal power returns to the baseline before the next injection. The stirring speed was 750 rpm. Couple of ITC binding isotherms for direct and reverse titrations were globally fitted to a 1:1 model using the AFFINImeter software thus yielding a single set of binding parameters per interaction.

Live cell imaging

Live-cell analysis was performed on a Deltavision Elite system using a × 40 oil objective with a numerical aperture of 1.35 (GE Healthcare). The DeltaVision Elite microscope was equipped with a CoolSNAP HQ2 camera (Photometrics). Cells were seeded in eight-well Ibidi dishes (Ibidi) and before filming, the media was changed to Leibovitz's L-15 (Life Technologies). Appropriate channels were recorded for the times indicated. For transient transfections, DNA constructs were transfected into HeLa cells using lipofectamine 2000 (Life Technologies) 24 h prior to analysis. The nuclear/cytoplasmic distribution of YFP-FoxO3 was analysed using SoftWoRx (GE Healthcare) software.

Immunoprecipitations and displacement assay

Hela-FRT cell lines stably expressing mCherry wt or control (3A) B56 inhibitor were transfected with YFP-B56α. Cells were arrested in Thymidine (2,5mM) for 24h and released into Nocodazole (200ng/ml) for 18h. The expression of the inhibitors was induced 24h prior of collection with the addition of 4 ng/ml Doxycycline. Cells were collected by mitotic shake-off and lyzed in low salt lysis buffer (50mM NaCl, 50mM Tris pH7.4, 1mM EDTA, 1mM DTT, 0,1% NP40) supplemented with protease and phosphatase inhibitors (Roche) for 25 min on ice. Lysates were cleared for 15 min at 20000 rcf and incubated with 20 μL pre-equalibrated GFP-trap beads (ChromoTek) for 45 min at 4°C. Following 3 washes with lysis buffer, the beads were eluted in 25 μL 2x loading buffer, boiled for 5 min and separated by SDS-PAGE or subjected to quantitative Mass spectrometry as described in the Label-free LC-MS/MS analysis section.

.

Cell cycle synchronization for G1/S and M phosphoproteomics analyses

Stable HeLa Flp-In cells expressing either wild-type or mutant B56 inhibitor were arrested in either G1/S or mitotic phase of the cell cycle. For G1/S arrest, cells were treated with 2 mM thymidine for 24 hours with the induction of wild-type or mutant B56 inhibitor for the last 12 hours using 10 ng/mL doxycycline. A mitotic arrest was achieved by treating the cells with 2 mM thymidine for 24 hours, followed by release into media containing 100 ng/ml nocodazole and 10 ng/ul doxycycline and collected by mitotic shake-off. Cells were washed once with PBS and snap-frozen. The amino acid sequence of the 4 X LxxIxE inhibitor is: TGSTGSTGSTGSTGSLPRSSTLPTIHEEEELSLCTGSTGSTGSTGSTGSLPRSSTLPTIHEEEELSLCTGSTGSTGSTGSTGSLPRSSTLPTIHEEEELSLCTGSTGSTGSTGSTGSLPRSSTLPTIHEEEELSLC. The corresponding 4 X control inhibitor sequence is: TGSTGSTGSTGSTGSLPRSSTAPTAHAEEELSLCTGSTGSTGSTGSTGSLPRSSTAPTAHAEEELSLCTGSTGSTGSTGSTGSLPRSSTAPTAHAEEELSLCTGSTGSTGSTGSTGSLPRSSTAPTAHAEEELSLC

Label-free LC-MS/MS analysis

Pull-downs were analyzed on a Q-Exactive Plus quadrupole Orbitrap mass spectrometer (ThermoScientific) equipped with an Easy-nLC 1000 (ThermoScientific) and nanospray source (ThermoScientific). Peptides were resuspended in 5% methanol / 1% formic acid and loaded on to a trap column (1 cm length, 100 μm inner diameter, ReproSil, C18 AQ 5 μm 120 Å pore (Dr. Maisch, Ammerbuch, Germany)) vented to waste via a micro-tee and eluted across a fritless analytical resolving column (35 cm length, 100 μm inner diameter, ReproSil, C18 AQ 3 μm 120 Å pore) pulled in-house (Sutter P-2000, Sutter Instruments, San Francisco, CA) with a 45 minute gradient of 5-30% LC-MS buffer B (LC-MS buffer A: 0.0625% formic acid, 3% ACN; LC-MS buffer B: 0.0625% formic acid, 95% ACN). The Q-Exactive Plus was set to perform an Orbitrap MS1 scan (R=70K; AGC target = 1e6) from 350 - 1500 m/z, followed by HCD MS2 spectra on the 10 most abundant precursor ions detected by Orbitrap scanning (R=17.5K; AGC target = 1e5; max ion time = 50ms) before repeating the cycle. Precursor ions were isolated for HCD by quadrupole isolation at width = 1 m/z and HCD fragmentation at 26 normalized collision energy (NCE). Charge state 2, 3, and 4 ions were selected for MS2. Precursor ions were added to a dynamic exclusion list +/- 20 ppm for 15 seconds. Raw data were searched using COMET (release version 2014.01) in high resolution mode (Eng et al., 2013) against a target-decoy (reversed) (Elias and Gygi, 2007)version of the human proteome sequence database (UniProt; downloaded 2/2013, 40482 entries of forward and reverse protein sequences) with a precursor mass tolerance of +/- 1 Da and a fragment ion mass tolerance of 0.02 Da, and requiring fully tryptic peptides (K, R; not preceding P) with up to three mis-cleavages. Static modifications included carbamidomethylcysteine and variable modifications included: oxidized methionine. Searches were filtered using orthogonal measures including mass measurement accuracy (+/- 3 ppm), Xcorr for charges from +2 through +4, and dCn targeting a <1% FDR at the peptide level. Quantification of LC-MS/MS spectra was performed using MassChroQ (Valot et al., 2011) and the iBAQ method (Schwanhäusser et al., 2011). Missing values were imputed from a normal distribution in Perseus to enable statistical analysis and visualization by volcano plot (Tyanova et al., 2016). Statistical analysis was carried out in Perseus by two-tailed Student's t-test.

Phosphoproteomics analysis

Cell pellets were lysed in ice-cold lysis buffer (8 M urea, 25 mM Tris-HCl pH 8.6, 150 mM NaCl, phosphatase inhibitors (2.5 mM beta-glycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM sodium molybdate) and protease inhibitors (1 mini-Complete EDTA-free tablet per 10 ml lysis buffer; Roche Life Sciences)) and sonicated three times for 15 sec each with intermittent cooling on ice. Lysates were centrifuged at 15,000 x g for 30 minutes at 4˚C. Supernatants were transferred to a new tube and the protein concentration was determined using a BCA assay (Pierce/ThermoFisher Scientific). Equal protein amounts are carried forward for analysis. For reduction, DTT was added to the lysates to a final concentration of 5 mM and incubated for 30 min at 55˚C. Afterwards, lysates were cooled to room temperate and alkylated with 15 mM iodoacetamide at room temperature for 45 min. The alkylation was then quenched by the addition of an additional 5 mM DTT. After 6-fold dilution with 25 mM Tris-HCl pH 8, the samples were digested overnight at 37˚C with 1:100 (w/w) trypsin. The next day, the digest was stopped by the addition of 0.25% TFA (final v/v), centrifuged at 3500 x g for 15 min at room temperature to pellet precipitated lipids, and peptides were desalted. Peptides were lyophilized and stored at -80˚C until further use.

Phosphopeptide purification was performed as previously described (Kettenbach and Gerber, 2011). Briefly, peptides were resuspended in 2 M lactic acid in 50% ACN ("binding solution"). Titanium dioxide microspheres were added and vortexed by affixing to the top of a vortex mixer on the highest speed setting at room temperature for 1 h. Afterwards, microspheres were washed twice with binding solution and three times with 50% ACN / 0.1% TFA. Peptides were eluted twice with 50 mM KH2PO4 (adjusted to pH 10 with ammonium hydroxide). Peptide elutions were combined, quenched with 50% ACN / 5% formic acid, dried and desalted.

Phosphopeptides were resuspended in 133 mM HEPES (SIGMA) pH 8.5, TMT reagent (ThermoFisher Scientific) stored in dry acetonitrile (ACN) (Burdick & Jackson) was added, vortexed to mix reagent and peptides. After 1 hr at room temperature, an aliquot from each channel was withdrawn to check for labeling efficiency while the remaining reaction was stored at -80°C. Once labeling efficiency was confirmed to be at least 95%, each reaction was quenched with ammonium bicarbonate for 10 minutes, mixed, acidified with 20% TFA, and desalted. The desalted multiplex was dried by vacuum centrifugation and separated by offline pentafluorophenyl (PFP)-based reversed phase HPLC fractionation as published (Grassetti et al., 2017). Briefly, TMT-labeled phosphopeptides were separated over a gradient of 5%-55% Buffer B from 0 to 61 mins. Forty-eight fractions were collected and concatenated into 24 by mixing the nth and nth+24th fraction. Buffer B: 95% ACN/0.1% TFA; Buffer A: 3% ACN/0.1% TFA

TMT-labeled samples were analyzed on an Orbitrap Fusion (Senko et al., 2013) mass spectrometer (ThermoScientific) equipped with an Easy-nLC 1000 (ThermoScientific). Peptides were resuspended in 8% methanol / 1% formic acid across a column (45 cm length, 100 μm inner diameter, ReproSil, C18 AQ 1.8 μm 120 Å pore) pulled in-house across a 2 hr gradient from 3% acetonitrile/0.0625% formic acid to 37% acetonitrile/0.0625% formic acid. The Orbitrap Fusion was operated in data-dependent, SPS-MS3 quantification mode (McAlister et al., 2014; Ting et al., 2011) wherein an Orbitrap MS1 scan was taken (scan range = 350 - 1200 m/z, R = 120K, AGC target = 3e5, max ion injection time = 100ms). Followed by data-dependent Orbitrap trap MS2 scans on the most abundant precursors for 3 seconds. Ion selection; charge state = 2: minimum intensity 2e5, precursor selection range 650-1200 m/z; charge state 3: minimum intensity 3e5, precursor selection range 525-1200 m/z; charge state 4 and 5: minimum intensity 5e5). Quadrupole isolation = 0.7 m/z, R = 30K, AGC target = 5e4, max ion injection time = 80ms, CID collision energy = 32%). Orbitrap MS3 scans for quantification (R = 50K, AGC target = 5e4, max ion injection time = 100ms, HCD collision energy = 65%, scan range = 110 - 750 m/z, synchronous precursors selected = 5). The raw data files were searched using COMET with a static mass of 229.162932 on peptide N-termini and lysines and 57.02146 Da on cysteines, and a variable mass of 15.99491 Da on methionines and 79.96633 Da on serines, threonines and tyrosines against the target-decoy version of the human proteome sequence database (UniProt; downloaded 2/2013, 40482 entries of forward and reverse protein sequences) and filtered to a <1% FDR at the peptide level. Quantification of LC-MS/MS spectra was performed using in house developed software. Phosphopeptide intensities were adjusted based on total TMT reporter ion intensity in each channel to adjust for mixing errors and log2 transformed. P-values were calculated using a two-tailed Student's t-test assuming unequal variance.

Inhibition of PP2A in lysates

Recombinant GST-Arpp19 wt or S62A mutant (235μg) was incubated with ~ 6 μg purified MASTL/Greatwall and thio-ATP (Tocris) in kinase buffer (50mM Tris-HCl pH 7.5, 10mM MgCl2, 0.1mM EDTA, 2mM DTT, 0.01% Brij 35) for 2h at 30°C. The GST Arpp19 proteins were concentrated and size-exclusion chromatography was performed on a Superdex 200 10/300 column and the buffer was exchanged to 300mM NaCl, 50mM Tris pH 8, 8,7 % glycerol. Peak fractions were collected, pooled and concentrated. Aliquots were snap frozen in liquid nitrogen.

Following double thymidine synchronization, cells were released into Nocodazole 200ng/ml for 16 hours. Mitotic Cells were collected by shake-off and counted. Lysates from 5x106 cells were analyzed per condition. Cells were lysed in lysis buffer (150mM NaCl, 50mM Tris pH 7,4, 0,1% NP40, 1mM DTT, supplemented with EDTA-free protease inhibitors (Roche)) plus 50 μg of the corresponding PP2A inhibitors. Full-length thio-phosphorylated Arpp19 WT or S62A was used for the inhibition of PP2A-B55. A high affinity LxxIxE peptide (WLPRSSTLPTIHEEEELSLC) or control peptide (WLPRSSTLPTAHADSVLSLC) were used to inhibit PP2A-B56 specifically. Cells were lyzed for 5 or 15 minutes at 30°C and shaking and the reactions were stopped by the addition of lysis buffer supplemented with 2xPhosStop tablets (Roche). Lysates were cleared for 15 minutes, 20000 rcf at 4°C and snap frozen in liquid nitrogen.

ARPP19 and B56 peptide lysate phosphoproteomics analysis

Lysates were precipitated by adding 4x volume of ice-cold acetone (Burdick & Jackson) and frozen for 1 h at -20°C. Afterward, samples were centrifuged at 14,800 x g for 15 min at 4°C to pellet precipitated proteins. Pellets were washed twice with ice-cold acetone and dried. Proteins were resuspended in urea lysis buffer as described above. Phosphopeptide enrichment was carried out using the Fe-NTA phosphopeptide enrichment kit (ThermoFisher) according to manufacturers instructions. Phosphopeptides were labeled with TMT reagents as described above, and offline separated. TMT-labeled samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer (ThermoScientific) equipped with an Easy-nLC 1200 (ThermoScientific). Peptides were resuspended in 8% methanol / 1% formic acid across a column (45 cm length, 100 μm inner diameter, ReproSil, C18 AQ 1.8 μm 120 Å pore) pulled in-house across a 2 hs gradient from 3% acetonitrile/0.0625% formic acid to 37% acetonitrile/0.0625% formic acid. The Orbitrap Lumos was operated in data-dependent, SPS-MS3 quantification mode (McAlister et al., 2014; Ting et al., 2011) wherein an Orbitrap MS1 scan was taken (scan range = 350 - 1250 m/z, R = 120K, AGC target = 2.5e5, max ion injection time = 50ms), followed by data-dependent Orbitrap MS2 scans on the most abundant precursors for 2 seconds. Ion selection; charge state = 2: minimum intensity 2e5, precursor selection range 650-1250 m/z; charge state 3: minimum intensity 3e5, precursor selection range 525-1250 m/z; charge state 4 and 5: minimum intensity 5e5). Quadrupole isolation = 1 m/z, R = 30K, AGC target = 5e4, max ion injection time = 55ms, CID collision energy = 35%). Orbitrap MS3 scans for quantification (R = 50K, AGC target = 5e4, max ion injection time = 100ms, HCD collision energy = 65%, scan range = 100 - 500 m/z, synchronous precursors selected = 5). The raw data files were searched and data was processed as described above.

In vitro phosphatase motif assay

293T Freestyle cells were transiently transfected with pEXPR-B56α, pCBS-KS+-2AAA, and pCBS-KS+PP2ACA. Forty-eight hours after transfection, cells were lysed in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, and 1 mM MnCl2), and sonicated three times for 15 sec each with intermittent cooling on ice. Lysates were centrifuged at 15,000 x g for 30 min at 4˚C. Strep-Tactin sepharose was added to the lysates and incubated while rotating for 2 h at 4˚C. Beads were collected by centrifugation, washed three times with lysis buffer, and eluted with 1x Buffer E (Strep-Tactin elution buffer with desthiobiotin). Libraries of naturally-occurring phosphopeptides were generated from 293T Freestyle cells. Cells were lysed in urea lysis buffer as described above with the modification that after reduction and alkylation, the lysate was diluted 2.5-fold, and the protease Lys-C was added to a final concentration of 1:100 w/w. After overnight digest, peptides were desalted, phosphopeptides were enriched, and labeled with TMT reagents as described above. Individual pools of TMT labeled phosphopeptides were dephosphorylated with PP2A-2AAA-B56α in the presence or absence of 10 nM Calyculin A for 0.5, 1, 2, 3, or 4 hs. Reactions were quenched with 0.1% TFA, mixed, desalted, dephosphorylated peptides were removed using TiO2 as described above, and analyzed on an Oribtrap Fusion Lumos mass spectrometer as described above.

In vitro phosphatase assays

In vitro phosphatase assays were performed with PP2A-B56α or PP2A-B55α, which were purified from HeLa cell extracts as described (Hein et al., 2017; Kruse et al., 2018). 15 μg of GST- fusion protein (GST-CDC20 49-78, engineered with different spacing or affinity of the LxxIxE motif) were incubated with CDK1-CyclinB1 (Sigma #SRP5009) in 50 μL reactions in kinase buffer (50 mM Tris-HCl pH 7,5, 10 mM MgCl2, 0,1 mM EDTA, 2 mM DTT, 0,01% Brij 35) with 500 μM ATP and 1μCi (γ-32P)-ATP (Perkin Elmer) at 30°C for 60 minutes. 15 Reactions were stopped by the addition of 10 μM RO-3306 (Calbiochem). GST-ADAM17 (V724-C827) was phosphorylated with recombinant Protein kinase A (New England Biolabs #P6000S). PD Spin Trap G25 columns (GE Healthcare) were used to exchange the buffer to phosphatase buffer (50 mM Tris pH 7,4, 1 mM MnCl2, 1mM DTT, 0,1% IGEPAL, 150mM NaCl).

Non-stick tubes were pre-treated with blocking buffer (50 mM Tris pH 7,4, 0,1 mM MnCl2, 1 mM MgCl2, 1 mM DTT, 0,1% NP40, 300mM NaCl, 2mg/ml BSA) on ice for the dephosphorylation reactions. 70 ng of PP2A-B56 holoenzyme was added to 84μL of phosphorylated substrate (~ 7μg). Samples of ~1,75 μg were taken out at the indicated time-points, added to 4x SDS loading buffer and boiled for 5 minutes. Samples were separated by SDS-PAGE. Gels were dried, exposed for 3 days and imaged on Typhoon FL 950 (GE Healthcare). Analyses and quantifications were carried out in ImageJ.

Michaelis-Menten kinetic parameters of the purified PP2A-B56αand PP2A-B55α holoenzymes were determined against the indicated phospho-peptides. Peptides were purchased from Peptide 2.0 Inc (Chantilly, VA, USA). The purity obtained in the synthesis was 98 % as determined by high performance liquid chromatography (HPLC) and subsequent analysis by mass spectrometry. Initial velocity (V0) was determined at varying concentrations of substrate (7.5 μM - 240 μM) incubated with ∼3 ng of the indicated PP2A holoenzyme in phosphatase buffer (50 mM Tris pH 7.4, 1 mM MnCl2, 1 mM DTT, 0.1% [vol/vol] IGEPAL, 150 mM NaCl) for 4 min at 30°C. Release of inorganic phosphate was measured using the PiColorLock Phosphate Detection System (Expedon). Data from three independent experiments were fitted to the Michaelis-Menten model and kinetic parameters extracted using GraphPad Prism version 6.0e for Mac OS X.

Data analysis

Phosphorylation site analysis were performed on phosphopeptides with the phosphorylation site localization score of 0.75 or higher. Icelogos were generated using singly phosphorylated sites with a phosphorylation localization score of 0.75 or higher (Colaert et al., 2009). For the network analysis, protein-protein interactions between previously identified B56 SLiM containing proteins (Hertz et al., 2016; Wu et al., 2017) and proteins with significantly increased phosphorylation sites (log2 ratio > 0.8 (1.75-fold), p-value < 0.05, phosphorylation site localization probability > 75%) were determined using the STRING database (Szklarczyk et al., 2017) and visualized in Cytoscape (Shannon et al., 2003).

Amphiregulin shedding assay

Amphiregulin shedding experiments were performed according to the manufactures protocol (R&D systems). To evaluate the effect of the ADAM17-B56 binding, we induced ADAM17 expression in the DLD-1 A17wt, I762A and LEE cell lines by changing to full medium containing 10 ng/ml Doxycycline (Sigma) and collected the supernatant after 24 h (constitutive shedding). For induced shedding the cells were treated with 600 µM H2O2 for 30 min (Merck), x-ray radiated with 10 Gy with a dose rate of 1 Gy/min using the x-ray generator CP160 (Faxitron X-Ray Corp.), or H2O vehicle control for 30 min and incubated for 3 h in full growth medium. Subsequently the medium was changed and the supernatant collected after 1h. The shedding results were normalized to the protein concentration to correct for possible differences in cell numbers.

Proliferation assay

DLD-1 A17wt, I762A and LEE cell lines were seeded at 1.5x 104 cells per well in a 96 well plate in 200 µl full medium supplemented with 10ng/ml Doxycycline. The cells were subsequently incubated at 37°C and 5% CO2 in an IncuCyte S3 (Sartorius) for 90 h and confluency was recorded every 2 h. The timepoint 50% confluence was calculated from growing curves, which were created using the Incucyte ZOOM software (Sartorius).

Invasion assay

Matrigel invasion assays were performed according to the manufacture's protocol. In short, ADAM17 expression was induced and upon re-hydration of the pre-coated matrigel invasion chambers (Corning), 2x 105 cells were seeded in 500 µl FBS free DMEM (Gibco) in the upper chamber of the inserts. Next, the inserts were transferred into a 24 well plate containing 1ml full DMEM and incubated for 24 h at 37°C. After incubation, invaded cells were fixed in 4% para-formaldehyde (Sigma), stained with 4% crystal violate (Sigma) and visualized under a light microscope (Axioplan 2, Zeiss). Invasion was determined by counting 10 randomly taken pictures (AxioCam, Zeiss) at a 10x magnification.

Cell surface biotinylation assay

ADAM17 expression in the DLD-1 A17wt, I762A and LEE cell lines was induced and 24h later, cells were washed twice in cold PBS and incubated for 30 min with 0.5 mg/ml non-cleavable EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) in PBS. The biotinylation was quenched by washing three times with 100mM glycine (AppliChem) in PBS, and additionally three PBS washes. Cells were lysed for 30 min in RIPA buffer supplemented with protease inhibitors. Cell lysates were cleared by centrifugation, protein concentration equalized by BCA assay, and supernatants incubated with streptavidin-agarose beads (Sigma-Aldrich) for 2 h at 4°C. Beads were washed three times in RIPA and bound proteins released by heating 5 min at 95°C in 2x SDS-PAGE sample buffer. Samples were analysed by western blot.

In vivo mouse model

Mice were randomly allocated into cages and mice within the same cage received the same treatment. On the day of injection, 4T1 A17wt, I761A and LEE cells were harvested and 1x104 cells in 50 µl PBS containing 10 ng/ml doxycycline injected into the fourth mammary fat pat of 7-weeks-old female BALB/c mice (Janvier labs). Sample size calculations performed with an alpha level of 0.05, 80%-90% power and an estimated difference between wt and LEE groups from previous experiments gave 8-11 mice/group. All mice were housed in ventilated cages in groups of 5 and maintained in a climate-controlled room at a temperature of 22 ± 2°C and a relative humidity of 50 ± 5% under a 12-hour light/dark cycle and fed a standard diet and water ad libitum. Measurements of the primary tumor size using calipers and the mouse weight were monitored in a blinded fashion 2-3 times a week. The mice received food and water ad libitum. The drinking water was supplemented with 1 mg/ml doxycycline (Sigma) and 5% sucrose (Sigma) and changed every 2-3 days. All experiments were performed in accordance with authorization and guidance from the Danish Inspectorate for Animal Experimentation. The cell lines were tested negative for murine pathogens by IMPACT testing (IDEXX Laboratories).

Quantification and statistical analysis

All statistical analysis was performed using GraphPad Prism version 6.0e for Mac OS X. Statistical details and definition of parameters can be found in figure legends. The statistical significance level was chosen as 0.05, but p-values for individual tests are indicated in the respective figure legends. Statistical methods were not employed to determine sample size or to determine whether the data met the assumptions of the statistical approach.

Data availability

Mass spectrometry data have been deposited to ProteomeXchange PXD015205, MassIVE MSV000084245, password p730.

Expanded view Figure legends