In Cellulo Kinase Screen Identifies Potential MASTL Substrates

doi:10.21203/rs.3.rs-1503557/v1

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In Cellulo Kinase Screen Identifies Potential MASTL Substrates

https://doi.org/10.21203/rs.3.rs-1503557/v1

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MASTL (microtubule-associated serine/threonine kinase-like) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate (in cellulo) kinase screen approach combined with stable isotope labelling of amino acids in cell culture to identify potential substrates and a consensus motif of MASTL. Using the related AGC kinase family members AKT1/2, the in cellulo assay identified several known and new substrates highly enriched with the validated consensus motif for AKT. Applying this method to MASTL identified 59 phospho-sites on 26 novel proteins significantly increased in the presence of active MASTL. Subsequent in vitro kinase assays confirmed that MASTL was capable of phosphorylating hnRNPM, YB1, RPS6, TUBA1C, and RPL36A under some conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.

MASTL

kinase

AKT

AGC kinase

SILAC

hnRNPM

MASTL (microtubule-associated serine/threonine kinase-like) is the human orthologue of Drosophila Greatwall (Gwl) kinase ^1,2, a protein essential for mitosis ³. Since its discovery, MASTL has become known as a master regulator of mitotic entry and integrity ^4–7. More recently, its roles have expanded to include meiosis ^8–10, recovery from DNA damage ^11–13, DNA replication ¹⁴, platelet maturation ^15,16, cell migration and actomyosin contraction ¹⁷. Unsurprisingly, these roles have led to MASTL being identified as a potential oncogenic kinase ¹⁸ with upregulation and over-expression correlating with poor patient outcomes in a variety of cancer types, including breast ^19–21, colon ^22,23 uterine ²², head and neck ²⁴ and pancreatic cancer ²⁵. Conversely, knockdown of MASTL has also been shown to sensitize cancer cells to a variety of DNA damaging therapies including radiotherapy ²⁶, cisplatin ^24,27 and gemcitabine ²⁵. Understanding the functions of MASTL and how these might promote cancer development and therapy resistance are of considerable importance and have driven recent effort towards the development of specific MASTL inhibitors ^28–31.

Currently the only validated substrate for MASTL is alpha-endosulﬁne (ENSA) and the highly related cAMP-regulated phosphoprotein 19 (Arpp-19). MASTL phosphorylates these proteins on a single site (S-67 and S-62 respectively) ^32,33, converting ENSA/Arpp-19 into an unfair competitive inhibitor of the phosphatase PP2A-B55 ³⁴. This inhibition is essential for correct mitotic entry and progression ^1,35, as it prevents PP2A-B55 from removing CDK1 phosphorylation events that drive correct mitotic entry and progression ^36,37. The MASTL/ENSA/PP2A axis is well conserved, with yeast (S. cerevisiae) Rim15 (MASTL) phosphorylating endosulfines Igo1 and Igo2, which in turn inhibit PP2ACdc55 ^38,39. Interestingly, during nutrient starvation, Rim15 phosphorylates additional substrates including the transcription factors Msn2p/4p and Hsf1p ⁴⁰. This raises the possibility that there are also additional substrates beyond ENSA/Arpp-19 for MASTL which might help explain the expanding array of functions attributed to MASTL. Several recent efforts have been made to identify potential MASTL substrates with conflicting results. Work by Bisteau et al ⁷, identified 101 mitotic proteins that were dephosphorylated upon MASTL knockout in immortalized mouse embryonic ﬁbroblasts (MEFs). Of these 6 were weakly phosphorylated in vitro by purified MASTL, but no decrease was observed using kinase dead MASTL, indicating that none are MASTL substrates ⁷. However, work by Hermida et al ⁴¹ identified 56 potential substrates phosphorylated by MASTL using a Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics approach (siKALIP), suggesting that MASTL may phosphorylate a wide variety of targets. Consequently, additional research on the potential substrates of MASTL is needed.

Of note MASTL is a member of the AGC kinase family that includes AKT and PKA, which each phosphorylate more than 130 and 370 substrates respectively ⁴². A notable characteristic of the AGC kinase family is that its members often phosphorylate the same substrates and residues. This is in part due to a shared consensus motif for AGC kinases, with a strong preference for basic residues Arginine (R) and Lysine (K) N-terminal of a Serine/Threonine (S/T) phospho-site ⁴². Currently, the consensus motif for MASTL substrates remains unknown, however, basic Lysine (K) residues are present N-terminal of the Serine phosphorylation site (-4 and -7 positions) in Arpp-19/ENSA, suggesting that MASTL may contain the same AGC family preference for basic residues.

Here we describe an unbiased whole cell lysate kinase screen approach that combines SILAC (in cellulo) to identify novel substrates of MASTL kinase. We validated the utility of the in cellulo approach using the related AGC family member AKT2, identifying known substrates and the kinase motif for AKT2. Using active recombinant MASTL we demonstrated its significant activity towards additional substrates using a peptide library array. Furthermore, using our in cellulo screen, we identified 26 novel substrates potentially phosphorylated by MASTL along with its preferred kinase motif. Subsequent in vitro kinase assay demonstrated that MASTL can phosphorylate YB1, RPS6, TUBA1C, RPL36A and hnRNPM, under certain conditions. Collectively, these data suggest that MASTL may phosphorylate additional substrates, which likely support its expanding functional repertoire in cancer progression and therapy resistance.

Validation of whole-cell lysate (in cellulo) kinase screen using AKT kinase

Identification of in vivo kinase-substrates has been a major stumbling block that has hampered our understanding of these important cellular enzymes. To help overcome this, we utilized a modified version of the previously reported whole-cell lysate (in cellulo) approach combined with stable isotope labelling with amino acids in cell culture (SILAC) ^43,44. This assay attempts to reconstitute the cellular environment by mixing an active recombinant kinase with a concentrated cellular lysate that has had its endogenous kinases irreversibly inhibited, allowing phosphorylation events on any potential cellular substrate to be enriched and identified by mass spectrometry. To validate the assay, we took advantage of the well characterized and related AGC kinase family member AKT, for which active kinase is commercially available. First, in vitro kinase assays were performed (Fig. 1A) without (Control) or with active (AK) or heat-deactivated (DK) AKT1 kinase. These were incubated with purified recombinant YB1, a known AKT1 substrate ^45,46. Reactions were stopped after 30- or 60-min by the addition of Laemmli buffer, and samples were then processed for western blot and LC-MS/MS. Importantly, western blots confirmed that AKT1 was phosphorylated on T-308, indicating the kinase was active, while heat-deactivation (DK) markedly reduced the levels of phosphorylation (Fig. 1B). Furthermore, phosphorylation of YB1 on S-102, was only detected by western blot when incubated with active kinase (AK), with no bands detected in DK or control lanes (Fig. 1C). Notably, peak phosphorylation occurred after a 60 min incubation, while ATP concentration did not notably impact phosphorylation levels. Mass spectrometry confirmed these results and identified additional phosphorylation events on T-80 and S-209 of YB1 (Fig. 1C, Supplementary Table S1), which have not previously been associated with AKT1 activity. These results confirmed that commercially purchased active AKT kinase could successfully phosphorylate a known AKT substrate in vitro.

We therefore utilized active recombinant AKT2 kinase to validate the ability of the in cellulo kinase assay to identify known and novel phosphorylation events. SILAC labelled MCF10A cells were cultured as previously described ¹⁹. Briefly, both heavy and light labelled cells were lysed and treated with the pan-kinase inhibitor fluorosulphonylbenzoladenosine (FSBA). Lysates were concentrated and supplemented with ATP, with or without recombinant active AKT2 kinase (Fig. 1D). Following protease digestion, samples were phospho-enriched using the EasyPhos method ⁴⁷ and analyzed on a Q Exactive HF mass spectrometer.

The in cellulo screen identified a total of 3035 phospho-sites on 1085 proteins of which 89 phospho-sites on 65 proteins (including AKT2) showed a Log₂ fold-change greater than 0.5, indicating potential phosphorylation by AKT2 (Fig. 1E and Supplementary Table S3). Of note there were an additional 54 known protein substrates of AKT that fell below the Log₂ fold-change significance threshold. Within the 65 high confidence proteins, 9 have previously been reported as AKT substrates of which 8 are validated AKT phosphorylation sites ⁴⁸. These included AKT1S1 (T-246), EDC3 (S-161), RPS6 (S-236, S-240), LMNA (S-407), TBC1D4 (S-341, S-588), and IRS2 (S-306). STRING and KEGG pathway analysis found that 51 of the 65 proteins were directly or indirectly linked to AKT1/2 (Fig. 1F) and were strongly enriched for proteins involved in ribosome and AMPK and mTOR signaling (Fig. 1G), in line with the reported downstream targets of the AKT pathway ⁴⁹. WebLogo alignment ⁵⁰ of the phosphorylated peptide sequences centered around the phosphorylated residue showed highly significant enrichment for serine phosphorylation with downstream enrichment of basophilic residues matching the published AKT phosphorylation motif (Fig. 1H). Taken together, these results indicate that the in cellulo kinase screen can identify known and potentially novel substrates along with the consensus phosphorylation motif for a well characterized AGC kinase family member.

MASTL in cellulo kinase screen

The above results indicated that our modified in cellulo kinase screen could be used to identify novel substrates of MASTL kinase. However, the screen requires high quality commercially available amounts of active MASTL kinase. We therefore partnered with Reaction Biology (previously ProQinase, Freiburg, Germany) to manufacture active human MASTL kinase. Briefly, GST-tagged full length wild-type human MASTL was expressed in Sf9 insect cells. Confluent cultures were treated with or without Okadaic Acid for 3 h and the soluble, active MASTL kinase purified by affinity chromatography (Supplementary Figure S1A). The activity and ability of MASTL kinase to phosphorylate substrates was then determined using a S/T/Y-Physiological Kinase Substrate Finder Assay (Reaction Biology). Briefly, in vitro radioactive ATP (P³³) based MASTL kinase assays were performed using a library of 720 peptides (13 amino acids long) derived from human protein sequences known to be phosphorylated in vivo. Over 150 peptides showed significant phosphorylation above autophosphorylation and background levels following incubation with active MASTL (Supplementary Figure S1B-C, Supplementary Table S3). This included MBP (myelin basic protein), which has previously been used as an in vitro substrate for MASTL ^51,52. Pathway analysis identified strong enrichment for proteins involved in MAPK and PI3K signaling (Supplementary Figure S1D-E), including EGFR and MYC, for which MASTL has been linked as an upstream regulator ^23,25. Taken together these results confirm that recombinant MASTL kinase possesses sufficient activity to be successfully used in vitro assays. In addition, MASTL is capable of phosphorylating peptide sequences beyond ENSA/Arpp-19.

Therefore, we next repeated the in cellulo kinase screen (Fig. 1D), substituting MASTL for AKT2. MCF10A cell lysates were again used as they express low levels of endogenous MASTL ¹⁹. A total of 1626 phospho-peptides were identified with 59 peptides showing a Log₂ fold increase greater than 0.5 (Fig. 2A, Supplementary Table S4). These 59 phospho-peptides mapped to 67 potential proteins, including exogenous active MASTL (Fig. 2B). The higher number of proteins was due to the homology of some peptides matching with multiple closely related protein variants (e.g., HIST1H1E, HIST1H1A, HIST1H1T etc.). Importantly, the top phospho-site identified was S-62/67 on ENSA/Arpp-19, providing an internal control for the in cellulo assay. STRING analysis of all potential phospho-peptides showed strong enrichment for proteins involved in regulation of mRNA translation, splicing, nuclear pore function cytoskeleton/migration and the expected PP2A phosphatase regulation. Alignment of phosphorylated peptide sequences, which showed a > 1.0 Log₂ fold increase, revealed an enrichment for serine phosphorylation with downstream enrichment of basophilic residues (K, R) from the − 3, to -7 positions, along with upstream polar residues from + 1 to + 4 and basophilic (K) residues from + 7 to + 9 (Fig. 2C). Notably, the top phosphorylated peptide (CACNA1C) from the S/T/Y-Physiological Kinase Substrate Finder Assay, showed some similarity to the MASTL consensus motif with enrichment of basic residues downstream and polar residues upstream of the potential serine phosphorylation site (Fig. 2D). In support, Hermida et al also identified a strong enrichment of polar and hydrophobic residues at the + 1 to + 4 positions and a basic residue at + 5 ⁴¹. Taken together, these data indicate a possible MASTL consensus motif with a basic residue at the − 6 and + 5 positions, hydrophobic at -2, and polar/hydrophobic from + 1 to + 4. In summary, the in cellulo assay results demonstrate that MASTL may phosphorylate several potential novel substrates and define a consensus kinase motif for MASTL.

In vitro kinase validation of MASTL substrates

Our next goal was to functionally characterize the candidate substrate proteins from the in cellulo screen. To further refine the list of potential substrates, we cross-referenced the phosphorylated proteins with our previously published list of proteins that co-immunoprecipitated (Co-IP) with MASTL from mitotic HeLa cell extracts⁵³ and MASTL substrates predicted by Hermida et al ⁴¹. There were 11 proteins that were present in all three datasets, an additional 11 common to our in cellulo assay and Hermida’s dataset, and a further 15 common to our in cellulo assay and our previous Co-IP (Fig. 3A, Supplementary Table S4). These 3 groups of 37 proteins were clustered around the regulation of cell migration, RNA splicing, RNA translation and chromatin condensation (Fig. 3B). We selected 6 proteins spanning these 3 groups (LMNA, YBX1, TUBA1C, NPM1, HNRNPM and ENSA) and 2 found only our in cellulo assay (LYRA, RPS6), for further validation by in vitro kinase assay. These were selected based on previously reported functions that correlated with any of the known roles for MASTL (e.g., mitosis, EMT, cancer), the availability of commercial recombinant purified protein and those with the highest Log₂ fold-change from our in cellulo assay. Before beginning more detailed functional analysis we confirmed the activity of MASTL towards ENSA under the same in vitro assay conditions used for AKT1 (Fig. 1A). We compared ENSA phospho-peptide intensities between ATP-only, active MASTL and heat-deactivated MASTL at different timepoints as a marker for specific kinase activity. Significant phosphorylation of ENSA on S-67 was only detected in presence of active kinase (AK), with peak phosphorylation observed in the 60 min reaction (Fig. 3C). Western blot analysis confirmed that active MASTL kinase (AK), significantly and specifically phosphorylated ENSA on S-67, with a 60 min incubation showing consistently higher levels of phosphorylation (Fig. 3D). Interestingly, multiple phospho-peptides from MASTL were also detectable in our mass spectrometry analysis. Of these, phosphorylation on T-194, which is essential for MASTL activity ⁵², was significantly more abundant in the active kinase sample compared to control, and importantly heat deactivation of MASTL (DK) reduced the abundance of T-194 and other phospho-sites (Fig. 3E). This, combined with specific phosphorylation on ENSA, confirmed that recombinant MASTL possessed sufficient activity under in vitro assay conditions.

The 7 remaining substrates were subjected to a single-shot in vitro kinase assay at 0.5- and 1.0-mM ATP for 60 min, and where sufficient substrate was available, 30 min reactions were also performed (Fig. 4, Supplementary Table S5). Strong and specific phosphorylation of ENSA was again observed (Fig. 4A), however no phosphorylation of LYRA was seen in any reaction. Similarly, while PTGES3 (p23) was phosphorylated, this was not specific and with phosphorylated residues detected under control (CTL) and dead kinase conditions (DK), indicating that neither LYRA nor p23 are likely substrates of MASTL. In contrast, a slight increased phosphorylation in the presence of the active kinase (AK) reactions was observed under some conditions for RPL36A and RPS6, while for hnRNPM, TUBA1C and YB1 more prominent increases in AK samples were present under multiple conditions (Fig. 4, blue rectangles). Notably, phosphorylation of hnRNPM on S-633 and S-48 on TUBA1C were also identified in the MASTL in cellulo screen (Fig. 2). While phosphorylation of YB1 on S-209 showed the strongest specificity, this site was not seen in our MASTL in cellulo assay but was found in our AKT1 screen (Fig. 1E). Based on these results, we selected hnRNPM as a potential novel substrate for further validation using more detailed in vitro kinase assays. Triplicate analysis identified 13 unique phospho-peptides that were present in at least 2 out of 3 replicates. Increased phosphorylation of Y-64 was observed at both 30 and 60 min, however, this is likely non-specific as MASTL is a serine/threonine directed kinase. Of the remaining sites only S-633/637 was significantly and specifically more abundant in the active kinase (AK) reaction (Fig. 5A, Supplementary Table S6). Interestingly, S-633 is located just downstream from the C-terminal RNA recognition motif (RRM) of hnRNPM (Fig. 5B) and shows some homology with our predicted MASTL consensus motif (Fig. 5B), with hydrophobic/polar residues enriched in + 1 to + 4 positions, and a basic residue at -6. Taken together, these results indicate that MASTL is capable of phosphorylating hnRNPM both in vitro and in cellulo.

Here we have presented a comprehensive analysis of MASTL substrates using a reconstituted and adapted cellular protein environment for use as a novel discovery tool. Our in cellulo kinase assay approach has successfully identified known and novel substrates for AKT2 and MASTL kinase. Analysis of substrate phosphorylation sites showed enrichment for kinase motifs that could aid in identifying targets for either kinase in future experiments. Our evidence suggests that MASTL may phosphorylate hnRNPM, TUBA1C, YB-1, and to a lesser extent RPS6 and RPL36A, in addition to ENSA/Arpp19. These findings may provide a greater understanding of the mechanistic links between MASTL and its biological and oncogenic functions.

MASTL has previously been shown to associate with microtubules during mitosis, decorating the mitotic spindle ¹. It co-precipitates with tubulin from mitotic cell extracts ¹⁹, which has previously been implicated as a potential MASTL substrate using a siKALIP approach ⁴¹. Combined with our observation here that MASTL can phosphorylate the core microtubule protein α-tubulin (TUBA1C) on multiple sites (T-41, S-48, and T-292) in cellulo, suggests that MASTL may regulate microtubule function. Notably, knockdown of MASTL results in multiple mitotic spindle and chromosome alignment defects ^1,2. Of note, the related AGC kinase, PKC, can also phosphorylate α-tubulin on S-165, stimulating microtubule dynamics ^54,55, and supporting the potential of AGC kinases as regulators of α-tubulin. Consequently, our results raise the possibility that MASTL could regulate spindle assembly by both maintaining CDK1 substrate phosphorylation, and by regulation of tubulin dynamics.

Our previous work identified several links between MASTL and epithelial mesenchymal transition (EMT), with MASTL over-expression overriding contact inhibition and disrupting cell migration in MCF10A breast epithelial cells. This corresponded with increased phosphorylation of several members of the β-catenin pathway and RPS6 ¹⁹. Interestingly, hnRNPM has been associated with increased activation of β-catenin signaling in breast cancer ⁵⁶, and increased EMT in breast ⁵⁷ and prostate cancer ⁵⁸. Both our results here and that of Hermida et al ⁴¹ identified the same S-633 phosphorylation site on hnRNPM, as a potential site phosphorylated by MASTL. Notably, the S-633 site on hnRNPM has been found in breast cancer and ischemic tumor tissues by mass spectrometry ^59,60, suggesting that this site is phosphorylated under certain conditions in vivo. However, no upstream kinase has been assigned for this phosphorylation site, nor has any functional significance been attributed. Of note, the phosphorylation site is located just next to the C-terminal RNA recognition motif, hence phosphorylation of S-633 could potentially regulate the binding of target RNA sequences, hnRNPM’s splicing activity, or impact binding of upstream regulatory proteins, such as AKAP8 ⁵⁷. Elucidating the links between hnRNPM and MASTL may give a greater understanding to the role MASTL plays in regulating EMT in breast cancer.

MASTL over-expression has also been linked with hyper-activation of the PI3K/AKT/mTOR pathway in humans ⁶¹, a function that may be related to its role in regulating nutrient response in yeast ^40,62. Interestingly, RPS6 plays an important role in regulating nutrient-mTORC1 signaling in human cells and is phosphorylated by multiple AGC kinases including AKT and p90RSK ^61,63. Similarly, we found that YB1, which also plays several key roles in the AKT pathway ⁴⁶, could be phosphorylated by both AKT1 and MASTL on S-209 in our in cellulo and in vitro screens and immunoprecipitates with MASTL ⁵³, suggesting this site may be phosphorylated by multiple AGC family members including MASTL. It will be of future interest to determine if phosphorylation by MASTL on either RPS6 and YB1 plays any role in their regulation and if this impacts nutrient response within mammalian cells.

In summary, here we show that MASTL has the potential to act like other AGC kinases and may phosphorylate several additional substrates in vitro in addition to Arpp-19/ENSA. Many of these new potential substrates, such as tubulin and hnRNPM, may help explain the growing list of MASTL functions including how MASTL promotes cancer, EMT and chemoresistance. Given MASTL’s rising role in cancer, there has been growing interest in the development of specific MASTL inhibitors ^28,30,31. We hope that our characterization of novel cellular targets and identification of a potential consensus motif for MASTL will aid their development and help improve next-generation inhibitors. Consequently, it will be of critical importance to further validate and confirm the physiological importance of these novel MASTL substrates in future research.

A full list of reagents used along with catalogue number and suppliers can be found in Supplementary Table S7.

Cell lines

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.1.5.3.30) 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.

In-gel digestion

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).

Arpp-19 – cAMP-regulated phosphoprotein 19

CDK1 – cyclin dependent kinase 1

EMT – epithelial mesenchymal transition

ENSA – alpha-endosulﬁne

FSBA – 5'-4-fluorosulphonylbenzoyladenosine

hnRNPM – heterogeneous nuclear ribonucleoprotein M

IAM - iodoacetamide

K - Lysine

MASTL – microtubule-associated serine/threonine kinase-like

PP2A – protein phosphatase 2A

PKA – protein kinase A

PKC – protein kinase C

R - Arginine

siKALIP - Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics

SILAC- Stable Isotope Labelling with Amino acids in Cell culture

S/T/Y – serine/threonine/tyrosine

TCEP-HCl – tris(2-carboxyethyl)phosphine hydrochloride

Acknowledgments: The Patricia Helen Guest Fellowship and Doherty Swinhoe Family Foundation for their generous support. The authors acknowledge the ANZAC Microscopy and Flow Facility, the Sydney Informatics Hub and the use of the University of Sydney’s high performance computing cluster, Artemis, and the mass spectrometry facility SydneyMS at the Charles Perkins Centre.

Author Contributions: KAM: Methodology, Validation, Formal analysis, Investigation, Data Curation, Visualization, Writing - Review & Editing. SR: Methodology, Validation, Formal analysis, Investigation, Writing - Review & Editing. RM: Validation, Investigation. BLP: Methodology, Resources, Supervision. DEJ: Methodology, Resources, Supervision. DNW: Resources, Supervision, Funding acquisition. AB: Conceptualization, Methodology, Validation, Formal analysis, Data Curation, Visualization, Writing - Original Draft, Funding acquisition, Supervision, Project administration

Data Availability Statement: All raw mass spectrometry data files, and MaxQuant and Proteome Discoverer output files have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository ⁶⁵ with the identifier PXD028678 (http://proteomecentral.proteomexchange.org).

Funding: This research was funded by Cancer Institute NSW (FRL/3-02), National Breast Cancer Foundation (IIRS-18-103) and Tour de Cure (RSP-230-2020).

Conflicts of Interest: The authors declare no conflict of interest”.

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No competing interests reported.

GraphicalAbstract.tif
SupplementaryFigureS1.tif
Supplementary Figure S1: MASTL S/T/Y-Physiological Kinase Substrate Finder Assay(A) Full-length human MASTL was expressed in Sf9 insect cells with or without 100 nM okadaic acid and purified using GSH-affinity (Glutathione) matrix. Purity was assessed samples by coomassie stained SDS-polyacrylamide gel electrophoresis. (B) Radiometric protein kinase assay (³³PanQinase® Activity Assay) was used for measuring the activity of MASTL on 720 biotinylated peptides known to be phosphorylated in vivo. The kinase assays were performed in 384-well polypropylene plates in a 50 μl reaction volume, the subsequent detection of phosphorylated, biotinylated peptides/proteins in 384-well, streptavidin-coated FlashPlate® HTS PLUS (Perkin Elmer, Boston, MA, USA). (C) The top 5 phosphorylated peptides from B are shown along with controls for Autophosphorylation (Auto; sample kinase, without substrate) background (BG; EDTA-inactivated MASTL, without substrate) across the two plates are shown separately. (D) SRING network and (E) KEGG pathway analysis of all peptides with >35 CPM from B.
SupplementaryTableS1AKT1YB1invitro.xlsx
Supplementary Table S1: AKT in vitro kinase assayLabel-free quantitation, Proteome Discoverer v2.3. Each master protein present in the reaction (AKT1 kinase and one substrate protein; blue highlight) is sub-divided into identified phospho-peptides (orange highlight) – Sub-tables can be expanded with (+) icon in left-hand margin. Column information includes accession numbers, %coverage, number of peptide spectral matches (#PSMs), Sequest HT score, KEGG pathways, abundance ratios and grouped abundances, missed cleavages, confidence scores, co-modifications, and peptide location within the master protein.
SupplementaryTableS2AKTSILAC.xlsx
Supplementary Table S2: AKT in cellulo kinase assaySummary excel file of phosphorylated peptides identified from the AKT2 in cellulo kinase screen (n=2). Data was analysed using MaxQuant (v.1.5.3.30) with integrated Andromeda search engine and Perseus.
SupplementaryTableS3ReactionBiology.xlsx
Supplementary Table S3: MASTL S/T/Y-Physiological Kinase Substrate Finder AssayExcel summary file containing the measured raw data (cpm) for the two 384-well plates labelled "A" and "B" used to measure the activity of purified MASTL kinase against a library of 720 biotinylated peptides using a radiometric protein kinase activity assay (33PanQinase® Activity Assay) using streptavidin-coated FlashPlate® HTS PLUS plates.
SupplementaryTableS4MASTLSILAC.xlsx
Supplementary Table S4: MASTL in cellulo kinase assaySummary excel file of phosphorylated peptides identified from the MASTL in cellulo kinase screen (n=2). Data was analysed using MaxQuant (v.1.5.3.30) with integrated Andromeda search engine and Perseus.
SupplementaryTableS5MASTLinvitroallsubs.xlsx
Supplementary Table S5: MASTL in vitro kinase assay screenLabel-free quantitation, Proteome Discoverer v2.3. Each substrate analysed separately and displayed in individual tabs. Each master protein present in the reaction (MASTL kinase and one substrate protein; blue highlight) is sub-divided into identified phospho-peptides (orange highlight) – Sub-tables can be expanded with (+) icon in left-hand margin. Column information includes accession numbers, %coverage, number of peptide spectral matches (#PSMs), Sequest HT score, KEGG pathways, abundance ratios and grouped abundances, missed cleavages, confidence scores, co-modifications, and peptide location within the master protein.
SupplementaryTableS6MASTLinvitrotriplicates.xlsx
Supplementary Table S6: In vitro kinase assay validation of ENSA and hnRNPMLabel-free quantitation, Proteome Discoverer v2.3. Each substrate analysed separately and displayed in individual tabs. Each master protein present in the reaction (MASTL kinase and ENSA or hnRNPM; blue highlight) is sub-divided into identified phospho-peptides (orange highlight) – Sub-tables can be expanded with (+) icon in left-hand margin. Column information includes accession numbers, %coverage, number of peptide spectral matches (#PSMs), Sequest HT score, KEGG pathways, abundance ratios and grouped abundances, missed cleavages, confidence scores, co-modifications, and peptide location within the master protein.
SupplementaryTableS7ListofReagents.xlsx
Supplementary Table S7: List of ReagentsA list of all reagents used in this study along with their catalogue number and supplier.

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In Cellulo Kinase Screen Identifies Potential MASTL Substrates

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Abstract

Figures

Introduction

Results

Validation of whole-cell lysate (in cellulo) kinase screen using AKT kinase

MASTL in cellulo kinase screen

In vitro kinase validation of MASTL substrates

Discussion

Materials And Methods

Cell lines

Proteins, antibodies and reagents

Whole cell lysate (in cellulo) Kinase Assay: Sample Preparation

MASTL whole cell lysate (in cellulo) Kinase Assay: Mass Spectrometry and Data Analysis

In vitro kinase assays

SDS-PAGE and western blotting

In-gel digestion

Mass spectrometry of in vitro kinase assay samples

Experimental Design and Statistical Rationale

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1