A full list of reagents used along with catalogue number and suppliers can be found in Supplementary Table S7.
MCF-10A breast cells were purchased from Sigma (#CLL1040) (38). MCF-10A cells were grown in DMEM/F12 (1:1) supplemented with 5% horse serum, 20 ng/mL recombinant human EGF, 0.5 ug/mL hydrocortisone, 100 ng/mL cholera toxin, and 10 ug/mL bovine insulin as previously described 19.
Proteins, antibodies and reagents
Active MASTL kinase (#1593-0000-2) and the Ser/Thr/Tyr-Physiological Kinase Substrate Finder Assay Service was purchased from and performed by Reaction Biology (previously ProQinase, Freiburg, Germany). Purified recombinant AKT2 was purchased from Active Motif (#81146). Purified recombinant active AKT1 (#ab62279) and recombinant proteins ENSA (#ab92932), YB1 (#ab187443), hnRNPM (#ab226407), p23 (#ab113183), LYAR (#ab163160), RPL36A (#ab159415), TUBA1C (#ab164660) were purchased from Abcam, and RPS6 (#H00006194-P01) was purchased from Abnova. Kinase reaction reagents included Tris-base, hydrochloric acid, magnesium chloride hexahydrate, DL-dithiothreitol and were purchased from Sigma, and adenosine 5’-triphosphate from New England Biolabs. Antibodies used for western blotting included anti-MASTL, phospho-YB1 (S-102), pan-AKT, phospho-AKT1 (T-308), phospho- Arpp-19/ENSA (S-62/67) (Cell Signaling Technology), as well as anti-YB1, anti-rabbit IgG HRP and anti-mouse IgG HRP (Abcam), and anti-His-ENSA 14. Reagents for mass spectrometry sample preparation including LC-MS grade water and formic acid were purchased from Pierce, HPLC grade trifluoroacetic acid and LiChrosolv acetonitrile from Sigma. Further details can be found in Supplementary Table S7.
Whole cell lysate (in cellulo) Kinase Assay: Sample Preparation
MCF-10A cells were labelled by stable isotope labelling of amino acids in cell culture (SILAC) as previously described 19. Briefly, cells were cultured for 6 population doublings in SILAC F12:DMEM (1:1) (Thermo Scientific) supplemented with either “heavy” lysine-13C614N2 (Lys8) and arginine-13C614N4 (Arg10), or the respective “light” counterparts (Lys0, Arg0). Labelled amino acid incorporation was checked routinely. Protein was harvested by scraping in 1 mL of ice-cold NP-40 buffer (1% NP-40, 150 mM NaCl, 50 mM Tris HCl pH 7.4, 10% Glycerol) plus protease inhibitor cocktail (Sigma) and PhosStop (Roche) and stored at -80 oC. 100 mM 5'-4-fluorosulphonylbenzoyladenosine (FSBA) in DMSO was slowly diluted 1:5 in pre-warmed NP-40 buffer at 37 oC. To irreversibly inhibit endogenous kinase activity, 1 mL of the FSBA solution was added to thawed lysates and shaken at 30 oC for 30 min at 1,200 rpm. Precipitated FSBA was removed by centrifugation and the supernatant was concentrated to 2.5 mL in a 10,000 MW centrifugal filter (Amicon). 1 mg of concentrated lysate was diluted 1:1 in 2x kinase assay buffer (50 mM Tris HCl pH 7.4, 10 mM MgCl2, 3 mM MnCl2, 3 µM Na3VO4, 2 mM DTT, and 500 µM ATP). 50 µg of recombinant purified MASTL kinase (Reaction Biology) or 10 µg AKT2 (Active Motif) was added to the heavy sample and incubated at 32 oC for 3 h under gentle agitation. The reaction was stopped by adding GdmCl buffer (6 M GdmCl, 100 mM Tris pH 8.5, 20 mM TCEP, 100 mM IAM) to each sample. Kinase reactions were heated to 95 oC for 5 min, cooled on ice, then kept at RT in the dark for 30 min to allow for peptide reduction and alkylation. Heavy and light samples were mixed 1:1 and 4x volumes of cold acetone were added to each mix and incubated at -20 oC overnight. Precipitated proteins were digested in Lys-C, and trypsin at 100:1 (w:w) overnight at 37 oC. The resultant mixture was then enriched for phosphorylated peptides using the EasyPhos method as described 47. Briefly, phospho-peptides were bound to TiO2 beads (5 µm Titansphere, GL Sciences) at a ratio of 10:1 (protein:beads) at 40 oC for 5 min. After washing (80% ACN, 0.5% 5% trifluoroacetic acid), phospho-peptides were eluted (40% ACN, 15% Ammonium Hydroxide), dried, and loaded onto in-house packed dual layer SDB-RPS stage tips (3M empore). The stage-tips were desalted and phospho-peptides eluted (80% ACN, 5% ammonium hydroxide), dried and stored at -80 oC until mass spectrometry analysis.
MASTL whole cell lysate (in cellulo) Kinase Assay: Mass Spectrometry and Data Analysis
Digested phospho-peptides were analysed as previously described. Briefly, a 50 cm x 75 µm fused silica column, packed in-house using 1.9 µM C18AQ particles using an Easy nLC-1200. Peptides were separated using a 195 min gradient using a binary buffer system of Buffer A (0.1% formic acid) and Buffer B (80% ACN, 0.1% formic acid) at a flow rate of 300 nL/min. Peptides were eluted with a gradient of 5–30% buffer B over 150 min, followed by 30–60% buffer B over 5 min, and 60–95% buffer B over 5 min. Peptides were analyzed on a Q-Exactive HF mass spectrometer operated in positive-ion DDA mode, with one full scan (300-1,650 m/z, R = 35,000 at 200 m/z), at a target of 3e6, selecting top 20 most abundant precursor ions (isolation window = 1.4 m/z, ion target 3e5) for HCD fragmentation (NCE = 27%) and MS2 scan (R = 35,000 at 200 m/z). Thermo RAW files were generated in centroid mode and analyzed using MaxQuant (v.188.8.131.52) with integrated Andromeda search engine, searching against the human whole proteome, with additions (Uniprot release 07.2018; 21,050 entries) Default MaxQuant settings were used, with 1% peptide spectral match (PSM) false discovery rate (FDR) followed by further filtering to 1% protein FDR. Precursor mass tolerance was set to 20 ppm for a first search followed by mass recalibration and 7 ppm tolerance for second search. Fragment ion tolerance was set to 7 ppm. The searches were conducted with carbamidomethylation of Cys set as a fixed modification. Phosphorylation of Serine, Threonine and Tyrosine, oxidation of Met and “light” Lysine and Arginine were replaced by “heavy” isotope-labelled amino acids 13C615N4-l-Arginine (Arg 10) and 13C615N2-l-Lysine (Lys 8) with Arginine and Lysine set as variable modifications. The match between runs and re-quantify options were enabled with matching elution time of 1.0 min. Bioinformatics were performed in Perseus (1.5), and Microsoft Excel (2010). Briefly, SILAC H/L ratios determined by MaxQuant were inverted for label-swap experiments, and Log2 transformed prior to statistical analysis. Non-significant Log2H/L ratios were eliminated using the Student’s t-test with Benjamini-Hochberg adjustment for multiple comparisons, with P < 0.05 as a significance cut off, ratios > 0.5 were considered significantly altered. KEGG pathway enrichments were determined from the parent protein identified from the individual phospho-peptides 64and interaction networks were produced using STRING v11.5 64.
In vitro kinase assays
In vitro kinase assay reaction mixtures (40 µl total) contained 500 ng of AKT1 or MASTL kinase and 100 ng of substrate protein in kinase reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 2 mM dithiothreitol, pH 7.4) in the presence of 0.5 mM or 1 mM ATP. Reactions were performed at 30 °C for 30 min and 60 min, then quenched with 2X SDS-polyacrylamide gel electrophoresis sample buffer (Bolt, Invitrogen).
SDS-PAGE and western blotting
Kinase reaction samples were heated for 10 min at 70 °C, then cooled on ice for 2 min. The full sample volume (less a small amount used for western blotting) was loaded onto Bolt 4–12% Bis-Tris Plus gels and run under reducing conditions with Bolt MES Running Buffer (Invitrogen) at 180–200 V. Prestained molecular weight markers were also included (SeeBlue Plus2, Invitrogen; GangNam-STAIN, iNtRON Biotechnology). Once kinase and substrate molecular weights were sufficiently separated, gels were washed and stained according to manufacturer’s instructions using SimplyBlue SafeStain (Invitrogen) before proceeding to in-gel digestion and mass spectrometry. For western blotting, gels were washed in water and proteins transferred onto PVDF membranes using the iBlot2 system (Invitrogen). Membranes to be probed for phospho-proteins were blocked in 5% bovine serum albumin (BSA; Sigma) in tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T), or otherwise in 5% low fat milk in TBS-T. Primary antibodies were diluted in 3% BSA/TBS-T and incubation was carried out overnight at 4 °C. Secondary antibodies were diluted in 5% LFM/TBS-T or 3% BSA/TBS-T (for phospho-proteins) and incubation was carried out for 1–2 h at room temperature. All membrane washes were carried out with TBS-T, except for the final wash before imaging, which used TBS. Protein bands were detected by chemiluminescence using Clarity Western ECL Substrate (Bio-Rad) and the ChemiDoc imaging system (Bio-Rad). Densitometry was analysed with ImageLab software.
Stained protein bands were excised with a clean scalpel and underwent destaining, reduction-alkylation and digestion using an In-Gel Tryptic Digestion Kit (Thermo Scientific). Manufacturer’s instructions were followed. Briefly, one volume of reagent was used per protein band, and trypsin digestion was carried out on a heat block overnight (16–18 h) at 37 °C.
Mass spectrometry of in vitro kinase assay samples
Digested samples were desalted, purified, and concentrated using C18 ZipTips (Z720070 (discontinued), Millipore), then dried by vacuum centrifugation and reconstituted in loading buffer (3% acetonitrile, 0.1% formic acid). Samples were injected onto a 50 cm x 75 µm internal diameter column (packed in-house with 1.9 µm Pur 120 C18 particles) using a Thermo Ultimate-3000 UHPLC. Peptides were separated using a 110 min gradient with a binary buffer system (buffer A = 0.1% formic acid; buffer B = 80% acetonitrile, 0.1% formic acid) at a constant flow rate of 0.250 µl/min. Peptides were eluted with a gradient of 5% buffer B over 30 min, then 5–10% buffer B over 1 min, then 10–35% buffer B over 59 min, then 35–95% buffer B over 3 min, then maintained at 95% buffer B for 4 min, then 95 − 5% buffer B over 1 min, then 5% buffer B maintained for a further 12 min. The column was maintained at 30 °C. Data-dependent acquisition (DDA) was performed with a Q Exactive Plus Orbitrap mass spectrometer operating in positive-ion mode, with a full-scan MS1 measured at 35,000 resolution (350 to 1550 m/z scan range; 50 ms injection time; 3x106 automated gain control (AGC) target) followed by isolation of up to 20 most abundant precursor ions for MS/MS (1.2 m/z isolation; 26 normalized collision energy; 17,500 resolution; 60 ms injection time; 3x105 AGC target). Thermo RAW files were generated in centroid mode. Raw data were processed with Proteome Discoverer v2.3 and v2.4 and searched with Sequest HT against the human UniProt database (07.2018; 21,050 entries). Full trypsin was set as the enzyme used with a maximum of 2 missed cleavages permitted. The mass tolerance for precursor ions was set to 10 ppm. The searches were conducted with carbamidomethylation on cysteine set as a static modification. Carbamidomethylation on histidine and lysine, phosphorylation of serine, threonine and tyrosine, deamidation of asparagine and glutamine, and oxidation of methionine were set as variable modifications. An automatic target FDR of 1–5% was set. Relative abundance levels of phosphorylation were found with label-free quantitation (LFQ) using the Minora Feature Detector.
Experimental Design and Statistical Rationale
For in cellulo kinase screening, two independent (n = 2) biological replicates were performed. Average Log2H/L ratios from the 2 replicates greater than > 0.5 were taken as potentially increased (phosphorylated) substrates for AKT and MASTL. Further stringency was applied using a Student’s t-test, with P < 0.05 taken as significant. For MASTL, substrates were further refined by cross-referencing with previously identified proteins that co-immunoprecipitated with MASTL from mitotic HeLa cell extracts 53. For the AKT1 + YB1 in vitro kinase assay, two (n = 2) biological replicates were performed, and for MASTL + ENSA and MASTL + hnRNPM in vitro kinase assays, three (n = 3) independent biological replicates were performed. Statistical significance for western blot was determined using 2-way ANOVA with Tukey’s multiple comparison’s test, and significance for mass spectrometry results was determined using an unpaired 1-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism software (v9.1.0).