Molecular Basis of the Mechanisms Controlling MASTL
2019; Elsevier BV; Volume: 19; Issue: 2 Linguagem: Inglês
10.1074/mcp.ra119.001879
ISSN1535-9484
AutoresDario Hermida, Gulnahar B. Mortuza, Anna-Kathrine Pedersen, Irina Pozdnyakova, Tam T. T. N. Nguyen, María Maroto, Michael R. Williamson, Tasja Wainani Ebersole, Giuseppe Cazzamali, Kasper D. Rand, Jesper V. Olsen, Marcos Malumbres, Guillermo Montoya,
Tópico(s)Bioinformatics and Genomic Networks
ResumoThe human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases. The human MASTL (Microtubule-associated serine/threonine kinase-like) gene encodes an essential protein in the cell cycle. MASTL is a key factor preventing early dephosphorylation of M-phase targets of Cdk1/CycB. Little is known about the mechanism of MASTL activation and regulation. MASTL contains a non-conserved insertion of 550 residues within its activation loop, splitting the kinase domain, and making it unique. Here, we show that this non-conserved middle region (NCMR) of the protein is crucial for target specificity and activity. We performed a phosphoproteomic assay with different MASTL constructs identifying key phosphorylation sites for its activation and determining whether they arise from autophosphorylation or exogenous kinases, thus generating an activation model. Hydrogen/deuterium exchange data complements this analysis revealing that the C-lobe in full-length MASTL forms a stable structure, whereas the N-lobe is dynamic and the NCMR and C-tail contain few localized regions with higher-order structure. Our results indicate that truncated versions of MASTL conserving a cryptic C-Lobe in the NCMR, display catalytic activity and different targets, thus establishing a possible link with truncated mutations observed in cancer-related databases. The gene encoding Microtubule-associated serine/threonine kinase-like (MASTL) 1The abbreviations used are:MASTLmicrotubule-associated serine/threonine kinase-likeGwlGreatwallLC-MS/MSliquid chromatography-tandem mass spectrometryHCDhigher-energy collisional dissociationHDX-MShydrogen/deuterium exchange mass spectrometry. 1The abbreviations used are:MASTLmicrotubule-associated serine/threonine kinase-likeGwlGreatwallLC-MS/MSliquid chromatography-tandem mass spectrometryHCDhigher-energy collisional dissociationHDX-MShydrogen/deuterium exchange mass spectrometry., also known as Greatwall (Gwl), was initially described in Drosophila as the Scant (Scott of the Antarctic) mutation (1White-Cooper H. Carmena M. Gonzalez C. Glover D.M. Mutations in new cell cycle genes that fail to complement a multiply mutant third chromosome of Drosophila.Genetics. 1996; 144: 1097-1111Crossref PubMed Scopus (37) Google Scholar). Soon after, Scant was shown to encode an essential protein kinase involved in mitosis (2Archambault V. Zhao X. White-Cooper H. Carpenter A.T. Glover D.M. Mutations in Drosophila Greatwall/Scant reveal its roles in mitosis and meiosis and interdependence with Polo kinase.PLoS Genet. 2007; 3: e200Crossref PubMed Scopus (88) Google Scholar) (3Yu J. Fleming S.L. Williams B. Williams E.V. Li Z. Somma P. Rieder C.L. Goldberg M.L. Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila.J. Cell Biol. 2004; 164: 487-492Crossref PubMed Scopus (125) Google Scholar). Flies lacking MASTL displayed an abnormal chromosome condensation and an impaired mitotic progression because of a delay in the late G2 phase to mitosis. Subsequent studies in Xenopus egg extracts revealed that MASTL was not only required for mitotic entry but also necessary for maintaining the CSF-arrested mitotic state in the extracts (4Yu J. Zhao Y. Li Z. Galas S. Goldberg M.L. Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts.Mol. Cell. 2006; 22: 83-91Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Based on these findings, MASTL was proposed to be involved in the Cdk1 autoregulatory loop, but its contribution remained unclear (4Yu J. Zhao Y. Li Z. Galas S. Goldberg M.L. Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts.Mol. Cell. 2006; 22: 83-91Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Later experiments revealed that MASTL's role in mitotic control, rather than targeting Cdk1 regulators, was to inhibit the activity of the PP2A-B55δ (5Vigneron S. Brioudes E. Burgess A. Labbé J.-C.C. Lorca T. Castro A. Greatwall maintains mitosis through regulation of PP2A.EMBO J. 2009; 28: 2786-2793Crossref PubMed Scopus (154) Google Scholar, 6Mochida S. Ikeo S. Gannon J. Hunt T. Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts.EMBO J. 2009; 28: 2777-2785Crossref PubMed Scopus (214) Google Scholar) phosphatase antagonising its activity. Intriguingly, none of the phosphatase subunits was targeted by MASTL, and the means to achieve inhibition remained vague. Nonetheless, the discovery of two related paralogs as substrates, Arpp19 and ENSA, which once phosphorylated by MASTL inhibit PP2A-B55δ, elucidated the kinase mechanism to inhibit PP2A (7Gharbi-Ayachi A. Labbé J.-C.C. Burgess A. Vigneron S. Strub J.-M.M. Brioudes E. Van-Dorsselaer A. Castro A. Lorca T. The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A.Science. 2010; 330: 1673-1677Crossref PubMed Scopus (305) Google Scholar, 8Mochida S. Maslen S.L. Skehel M. Hunt T. Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis.Science. 2010; 330: 1670-1673Crossref PubMed Scopus (313) Google Scholar). Remarkably, the inhibition was restricted to the PP2A-B55δ phosphatase complex, and other PP2A holoenzymes remained unaffected (8Mochida S. Maslen S.L. Skehel M. Hunt T. Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis.Science. 2010; 330: 1670-1673Crossref PubMed Scopus (313) Google Scholar). Accordingly, the importance of the precise activation and inactivation of MASTL for proper mitotic progression was highlighted. Aside from this well-conserved role in cell cycle regulation, recent reports have associated MASTL with the control of DNA replication through ENSA (9Charrasse S. Gharbi-Ayachi A. Burgess A. Vera J. Hached K. Raynaud P. Schwob E. Lorca T. Castro A. Ensa controls S-phase length by modulating Treslin levels.Nat. Commun. 2017; 8: 206Crossref PubMed Scopus (38) Google Scholar) and coordination during recovery from DNA damage (10Peng A. Yamamoto T.M. Goldberg M.L. Maller J.L. A novel role for greatwall kinase in recovery from DNA damage.Cell Cycle. 2010; 9: 4364-4369Crossref PubMed Scopus (39) Google Scholar, 11Peng A. Wang L. Fisher L.A. Greatwall and Polo-like kinase 1 coordinate to promote checkpoint recovery.J. Biol. Chem. 2011; 286: 28996-29004Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 12Wong P.Y. Ma HT. Lee H-j.J. Poon R.Y. MASTL(Greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation.Sci. Reports. 2016; 6: 22230PubMed Google Scholar), and a recent study of a MASTL thrombocytopenia-associated mutation has suggested a possible function for MASTL in regulating actin and cytoskeleton (13Hurtado B. Trakala M. Ximenez-Embun P. El Bakkali A. Partida D. Sanz-Castillo B. Alvarez-Fernandez M. Maroto M. Sanchez-Martinez R. Martinez L. Munoz J. Garcia de Frutos P. Malumbres M. Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeletal dynamics in platelets.J. Clin. Invest. 2018; 128: 5351-5367Crossref PubMed Scopus (16) Google Scholar). However, the link between MASTL and other pathways remains elusive, most likely because of the absence of additional well-known substrates besides Arpp19/ENSA. microtubule-associated serine/threonine kinase-like Greatwall liquid chromatography-tandem mass spectrometry higher-energy collisional dissociation hydrogen/deuterium exchange mass spectrometry. microtubule-associated serine/threonine kinase-like Greatwall liquid chromatography-tandem mass spectrometry higher-energy collisional dissociation hydrogen/deuterium exchange mass spectrometry. MASTL is classified as a member of the MAST subfamily of AGC kinases (14Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6217) Google Scholar). Closely related MASTL/Greatwall kinases are also present in other insects, vertebrates and yeast. Interestingly, MASTL contains a unique long insertion of about 550 non-conserved amino acids between the kinase subdomains VII and VIII, which is the typical location of the activation loop (3Yu J. Fleming S.L. Williams B. Williams E.V. Li Z. Somma P. Rieder C.L. Goldberg M.L. Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila.J. Cell Biol. 2004; 164: 487-492Crossref PubMed Scopus (125) Google Scholar, 15Vigneron S. Gharbi-Ayachi A. Raymond A.-A.A. Burgess A. Labbé J.-C.C. Labesse G. Monsarrat B. Lorca T. Castro A. Characterization of the mechanisms controlling Greatwall activity.Mol. Cell. Biol. 2011; 31: 2262-2275Crossref PubMed Scopus (55) Google Scholar). This segment has been termed the non-conserved middle region (NCMR) (16Blake-Hodek K.A. Williams B.C. Zhao Y. Castilho P.V. Chen W. Mao Y. Yamamoto T.M. Goldberg M.L. Determinants for activation of the atypical AGC kinase Greatwall during M phase entry.Mol. Cell. Biol. 2012; 32: 1337-1353Crossref PubMed Scopus (66) Google Scholar) because of the low conservation between orthologues and paralogues. MASTL activation also differs from that of most other AGC kinases, which encompasses the phosphorylation in three conserved regulatory motifs (17Kannan N. Haste N. Taylor S.S. Neuwald A.F. The hallmark of AGC kinase functional divergence is its C-terminal tail, a cis-acting regulatory module.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1272-1277Crossref PubMed Scopus (176) Google Scholar, 18Pearce L.R. Komander D. Alessi D.R. The nuts and bolts of AGC protein kinases.Nat. Rev. Mol. Cell Biol. 2010; 11: 9-22Crossref PubMed Scopus (980) Google Scholar) (1) in the activation segment, containing the T-loop (19Biondi R.M. Kieloch A. Currie R.A. Deak M. The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB.EMBO J. 2001; 20: 4380-4390Crossref PubMed Scopus (305) Google Scholar), which is usually phosphorylated by another AGC kinase member (20Mora A. Komander D. van Aalten D.M. Alessi D.R. PDK1, the master regulator of AGC kinase signal transduction.Sem. Cell Developmental Biol. 2004; 15: 161-170Crossref PubMed Scopus (664) Google Scholar), (2) in the hydrophobic motif (21Yang J. Cron P. Thompson V. Good V.M. Hess D. Hemmings B.A. Barford D. Molecular mechanism for the regulation of protein kinase B/Akt by hydrophobic motif phosphorylation.Mol. Cell. 2002; 9: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar) and (3) the tail linker motif (22Hauge C. Antal T.L. Hirschberg D. Doehn U. Thorup K. Idrissova L. Hansen K. Jensen O.N. Jørgensen T.J. Biondi R.M. Frödin M. Mechanism for activation of the growth factor-activated AGC kinases by turn motif phosphorylation.EMBO J. 2007; 26: 2251-2261Crossref PubMed Scopus (81) Google Scholar) (supplemental Fig. S1). The NCMR is phosphorylated (3Yu J. Fleming S.L. Williams B. Williams E.V. Li Z. Somma P. Rieder C.L. Goldberg M.L. Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila.J. Cell Biol. 2004; 164: 487-492Crossref PubMed Scopus (125) Google Scholar, 15Vigneron S. Gharbi-Ayachi A. Raymond A.-A.A. Burgess A. Labbé J.-C.C. Labesse G. Monsarrat B. Lorca T. Castro A. Characterization of the mechanisms controlling Greatwall activity.Mol. Cell. Biol. 2011; 31: 2262-2275Crossref PubMed Scopus (55) Google Scholar, 16Blake-Hodek K.A. Williams B.C. Zhao Y. Castilho P.V. Chen W. Mao Y. Yamamoto T.M. Goldberg M.L. Determinants for activation of the atypical AGC kinase Greatwall during M phase entry.Mol. Cell. Biol. 2012; 32: 1337-1353Crossref PubMed Scopus (66) Google Scholar), and some of the phosphorylation sites within the NCMR seem to be necessary for MASTL activity, and they have been shown to play a role in its localization (23Alvarez-Fernandez M. Sanchez-Martinez R. Sanz-Castillo B. Gan P.P. Sanz-Flores M. Trakala M. Ruiz-Torres M. Lorca T. Castro A. Malumbres M. Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 17374-17379Crossref PubMed Scopus (82) Google Scholar, 24Wang P. Galan J.A. Normandin K. Bonneil E. Hickson G.R. Roux P.P. Thibault P. Archambault V. Cell cycle regulation of Greatwall kinase nuclear localization facilitates mitotic progression.J. Cell Biol. 2013; 202: 277-293Crossref PubMed Scopus (34) Google Scholar, 25Wang P. Larouche M. Normandin K. Kachaner D. Mehsen H. Emery G. Archambault V. Spatial regulation of greatwall by Cdk1 and PP2A-Tws in the cell cycle.Cell Cycle. 2016; 15: 528-539Crossref PubMed Scopus (15) Google Scholar). However, it appears that no sequence within the NCMR is indispensable (15Vigneron S. Gharbi-Ayachi A. Raymond A.-A.A. Burgess A. Labbé J.-C.C. Labesse G. Monsarrat B. Lorca T. Castro A. Characterization of the mechanisms controlling Greatwall activity.Mol. Cell. Biol. 2011; 31: 2262-2275Crossref PubMed Scopus (55) Google Scholar, 16Blake-Hodek K.A. Williams B.C. Zhao Y. Castilho P.V. Chen W. Mao Y. Yamamoto T.M. Goldberg M.L. Determinants for activation of the atypical AGC kinase Greatwall during M phase entry.Mol. Cell. Biol. 2012; 32: 1337-1353Crossref PubMed Scopus (66) Google Scholar). Therefore, it remains unclear whether MASTL requires an activation loop phosphorylation for its regulation (15Vigneron S. Gharbi-Ayachi A. Raymond A.-A.A. Burgess A. Labbé J.-C.C. Labesse G. Monsarrat B. Lorca T. Castro A. Characterization of the mechanisms controlling Greatwall activity.Mol. Cell. Biol. 2011; 31: 2262-2275Crossref PubMed Scopus (55) Google Scholar) or substrate selection (16Blake-Hodek K.A. Williams B.C. Zhao Y. Castilho P.V. Chen W. Mao Y. Yamamoto T.M. Goldberg M.L. Determinants for activation of the atypical AGC kinase Greatwall during M phase entry.Mol. Cell. Biol. 2012; 32: 1337-1353Crossref PubMed Scopus (66) Google Scholar). On the other hand, MASTL holds a short AGC C-tail, which does not include the hydrophobic motif present in most AGC kinases (15Vigneron S. Gharbi-Ayachi A. Raymond A.-A.A. Burgess A. Labbé J.-C.C. Labesse G. Monsarrat B. Lorca T. Castro A. Characterization of the mechanisms controlling Greatwall activity.Mol. Cell. Biol. 2011; 31: 2262-2275Crossref PubMed Scopus (55) Google Scholar) (supplemental Fig. S1). Although some studies have addressed MASTL activation (15Vigneron S. Gharbi-Ayachi A. Raymond A.-A.A. Burgess A. Labbé J.-C.C. Labesse G. Monsarrat B. Lorca T. Castro A. Characterization of the mechanisms controlling Greatwall activity.Mol. Cell. Biol. 2011; 31: 2262-2275Crossref PubMed Scopus (55) Google Scholar, 16Blake-Hodek K.A. Williams B.C. Zhao Y. Castilho P.V. Chen W. Mao Y. Yamamoto T.M. Goldberg M.L. Determinants for activation of the atypical AGC kinase Greatwall during M phase entry.Mol. Cell. Biol. 2012; 32: 1337-1353Crossref PubMed Scopus (66) Google Scholar) and structural details in the absence of the NCMR region are available (26Ocasio C.A. Rajasekaran M.B. Walker S. Le Grand D. Spencer J. Pearl F.M. Ward S.E. Savic V. Pearl L.H. Hochegger H. Oliver A.W. A first generation inhibitor of human Greatwall kinase, enabled by structural and functional characterisation of a minimal kinase domain construct.Oncotarget. 2016; 7: 71182-71197Crossref PubMed Scopus (21) Google Scholar); the kinase activation mechanism and the role of the NCMR remain unclear. In this manuscript, we perform a combined analysis indicating that the NCMR region is essential for MASTL specificity and allosterically regulates the enzyme catalysis. In addition, we show that truncated products of MASTL may be catalytically active, using a cryptic C-lobe contained in a section of the NCMR, and display a different protein target palette in a HEK293 cell extract. Finally, hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveal MASTL dynamics. Full-length MASTL (H. sapiens) was obtained from a human cDNA library (Marcos Malumbres, CNIO, Spain). The DNA sequence corresponds to the serine/threonine-protein kinase Greatwall isoform 1 (Mammalian Gene Collection - MGC ID BC009107). The DNA sequence encoding for the Bonsai construct was obtained as a codon-optimized DNA sequence (Life Technologies). MASTL constructs were amplified by PCR using the LIC-MASTL primers. PCR products were used to clone the genes into the protein expression vector using Ligation Independent Cloning (LIC). A modified version of the commercially available pCEP4 (Invitrogen) expression plasmid containing LIC compatible overhangs, was used for protein overexpression. The resulting MASTL recombinant proteins contained an N-terminal tag coding for a 6xHis tag, a Twin-Strep-tag and TEV protease site (supplemental Table S1). Mutations into these constructs were achieved using the Quick-Change II Site-Directed Mutagenesis Kit (Agilent Technologies). All the constructs have been confirmed by full sequencing. Details of the cloning will be provided upon request. Human Embryonic Kidney EBNA 6E cell lines (HEK293 6E) were cultivated in Freestyle 293 F17 expression medium (Invitrogen) supplemented with 1% fetal bovine serum (FBS). One day before transfection, HEK293 6E cells were resuspended in fresh Freestyle 293 F17 expression medium to a cell density of 1.2 × 106 cells/ml and incubated at 37 °C overnight. Approximately, 15 mins before transfection, cells were resuspended in fresh non-supplemented Freestyle 293 F17 expression medium at a cell density of 20 × 106 cells/ml and incubated in the orbital shaker incubator at 37 °C, 70% humidity, 5% CO2 and 120 rpm (Ø50 mm), until being transfected. GigaPrep (Qiagen, Germany) plasmid DNA (50 μg/ml final) and Polyethylenimine "MAX" (PEI) (Polysciences) (100 μg/ml solution final) were directly added to the cell suspension. Complete Freestyle 293 F17 expression medium (1% FBS) was added to a final volume of 3L of cell suspension, 4 h post-transfection. Three days post-transfection the pellets were collected by centrifugation at 750 rpm for 10 mins at 4 °C. Cellular pellets of HEK293 6E cells overexpressing either MASTL FL or Bonsai were resuspended in Lysis buffer (50 mm Tris pH 8.0, 200 mm NaCl, 0.5 mm TCEP, 1 mm EDTA, 50 U/ml Benzonase/50 ml, 2x Tablets Complete Inhibitor mixture EDTA Free (Roche, Switzerland), 0.5% Triton X 10). After disruption by high-pressure EmulsiFlex-C3 Homogenizer (Avestin, Canada), cell debris and insoluble particles were removed by centrifugation at 10,000 × g at 4 °C. The supernatant was loaded onto a StrepTrap HP column (GE Healthcare) equilibrated in buffer A (50 mm Tris pH 8.0, 200 mm NaCl, 0.5 mm TCEP, 1 mm EDTA). After sample loading conclusion, the column was washed with 20 column volumes (CV) of buffer A. Elution of the protein was achieved by a single step elution with buffer B (buffer A + 2.5 mm Desthiobiotin). Enriched protein fractions were pooled together and incubated with ATP (300 μm final concentration) for 4 h at 4 °C. Soon after ATP-incubation, the sample was loaded onto a HisTrap HP column (GE Healthcare) equilibrated with buffer C (20 mm Tris pH 8.0, 200 mm NaCl, 0.5 mm TCEP, 2 mm MgCl2, 100 μm ATP). The column was washed first with 5–10 CV of buffer C, to be then further washed with 10 CV of buffer D (buffer C without ATP). Protein elution was achieved in a single step elution with buffer E (buffer D + 500 mm Imidazole). Protein-rich fractions were collected and dialyzed overnight on a 10 kDa MWCO SkaneSkin Dialysis Tubing (Thermo Scientific) with buffer D at 4 °C. The sample was concentrated (using a 10 kDa MWCO Centriprep Amicon Ultra devices) and if required loaded onto an S200–10/300GL column (GE Healthcare) equilibrated in buffer D. The protein peaks were concentrated (using a 10 kDa MWCO Centriprep Amicon Ultra devices), directly used for experiments or flash-frozen in liquid nitrogen and stored at - 80 °C. The protein concentration was determined using the theoretical molecular extinction coefficient at 280 nm calculated from the amino acid composition. The same purification procedure was used to purify all MASTL FL and Bonsai mutants. Resuspension and supernatant preparation of cellular pellets of HEK293 6E cells overexpressing MASTL450 were conducted as previously described for cellular pellets overexpressing MASTL FL and Bonsai, but the lysis buffer contained additionally 10% Glycerol (v/v). The supernatant was loaded onto a StrepTrap HP column (GE Healthcare) equilibrated in buffer A1 (buffer A + 10% Glycerol (v/v)). After sample loading conclusion, the column was washed with 20 column volumes (CV) of buffer A1. A single step elution was achieved with buffer B1 (buffer B + 10% Glycerol (v/v)). Enriched protein fractions were run on an SDS-PAGE gel (NuPAGE® 4–12% Bis-Tris Gel, Invitrogen) to be further stained with the Colloidal Stain Kit (Invitrogen). After extensive de-staining of the gel with ddH2O, the SDS-gel was digitalized using an Epson Perfection V750 Pro scanner. ImageQuant TL software (GE Healthcare) was used to perform a 1D gel analysis from the one-dimensional electroporation gel containing the samples. The protein concentration of the MASTL450 present in the sample was calculated using the percentage factor obtained from the 1D gel analysis and the total protein concentration of the sample determined by 280 nm absorbance (A280) measurement. Standard kinase assays were performed for 30 min at 30 °C in 10 μl of Kinase buffer (10 mm Tris pH 7.5, 50 mm KCl, 10 mm MgCl2, 1 mm DTT) supplemented with 50 μm cold ATP, 1.5 μCi [γ-32P] ATP (3000Ci/mmol) and 0.5 μm MASTL proteins prepared from the human HEK293 6E cells. In early experiments, either 50 μm of MBP (Merck Millipore, Catalogue #13–110) was added to the reaction mixture as a model kinase substrate or 50 μm recombinant full-length human Arpp19 protein (cAMP-regulated phosphoprotein 19 - UniProtKB - P56211) as verified MASTL substrate (7Gharbi-Ayachi A. Labbé J.-C.C. Burgess A. Vigneron S. Strub J.-M.M. Brioudes E. Van-Dorsselaer A. Castro A. Lorca T. The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A.Science. 2010; 330: 1673-1677Crossref PubMed Scopus (305) Google Scholar, 8Mochida S. Maslen S.L. Skehel M. Hunt T. Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis.Science. 2010; 330: 1670-1673Crossref PubMed Scopus (313) Google Scholar) (supplemental Table S1). The kinase reaction was concluded by the addition of LDS Sample Buffer to be further fractionated by SDS-polyacrylamide gel electrophoresis. Radioactive MASTL and substrate bands were identified by autoradiography, quantification of the signal was achieved by densitometry analysis of the autoradiograms using the ImageStudioLite 5.2.5 Software (Li-Cor Biosciences). Finally, the activity levels were corrected by the amount of protein present at each sample and represented as a percentage of the phosphorylation compared with that of the MASTL FL form. For the kinetic characterization of the MASTL constructs, a constant MASTL concentration was maintained (0.25 or 0.5 μm) while increasing the concentration of Arpp19 (1 - 240 μm). The length of the time courses was set so that the initial velocity at each substrate concentration presented a linear increase. Quantification of the signals as previously described. Data analysis was achieved using the Prism 6 software (Graphpad). In later experiments, 50 μm of several MASTL protein targets identified by our mass spectrometry experiments were used in the in vitro kinase assays. The fragments (24RGRGRPRKQPPVSPGTALVGSQKEPSEVPTPKRPRGRPKGS64 and 67KGAAKTRKTTTTPGRKPRGRPKKL EKEEEEGISQESSEEEQ107) of the human High mobility group protein HMG-I/HMG-Y (HMGA1 - UniProtKB P17096), the (16IKNSSVPRRTLKMIQPSASGSLVGRENELSAGLSKRKHRND56) of the human Geminin (GMNN - UniProtKB O75496) and the (42KPGGSDFLRKRLQKGQKYFDSGDYNMAKAKMKNKQLPTAAP82) of the human cAMP-regulated phosphoprotein 19 (Arpp19 - UniProtKB P56211) were fused to N-terminal tag containing a 6xHis tag, a LSL-tag (27Angulo I. Acebron I. de las Rivas B. Munoz R. Rodriguez-Crespo I. Menendez M. Garcia P. Tateno H. Goldstein I.J. Perez-Agote B. Mancheno J.M. High-resolution structural insights on the sugar-recognition and fusion tag properties of a versatile beta-trefoil lectin domain from the mushroom Laetiporus sulphureus.Glycobiology. 2011; 21: 1349-1361Crossref PubMed Scopus (30) Google Scholar) and TEV protease site. Quantification of the signals as previously described. MASTL wild-type colorectal cancer cell line DLD1 and heterozygous MASTL (+/p.K391fs*12) gastric cancer cell line 23132/87 were obtained from ATCC. DLD1 cells were cultured with DMEM low-glucose (Lonza, Switzerland), and 23132/87 cells with RPMI 1640 (Sigma-Aldrich) and were supplemented with 10% FBS (HyClone) filtered (0.2 μm), 0.1% Gentamicin Sulfate 50 mg/ml (Solmeglas S.L., Spain) and incubated at 37 °C in a humidified 5% CO2 atmosphere. Protein immunoprecipitation was performed with an in-house generated rat monoclonal antibody designed against the polypeptide comprising R22-S244 residues of mouse MASTL crosslinked with Protein A (Invitrogen) magnetic beads. Cell extracts were lysed in cold ELB lysis buffer (50 mm Hepes pH 7.5, 150 mm NaCl, 5 mm EDTA, 1% NP-40) with phosphatase and protease inhibitors, with rotatory agitation for 30 min, and then centrifuged at 13,000 rpm, 4 °C for 10 min. Protein concentration determination was performed for the supernatants with a BCA assay (Pierce). For protein immunoprecipitation, a crosslinked antibody was added to the protein in a rate of 2 μg/1 mg, and IP incubation was performed in a cold room at 4 °C with rotatory agitation for 16 h. After subsequent wash-outs, the IP sample was mixed with sample buffer (350 mm Tris-HCl pH 6.8, 30% glycerol, 10% SDS, 0.6 m DTT, 0.01% Bromphenol blue) and boiled for 5 min before loading in the electrophoresis gel. Membranes were incubated with a mouse monoclonal MASTL C-lobe antibody (Monoclonal 4F9 Millipore MABT372) and with a different clone of the rat monoclonal antibodies against MASTL N-terminal region. Antibodies for immunoblot were diluted 1:1000 in 3% BSA 0.05% PBS-Tween20. Proteins were transferred to nitrocellulose membranes which were incubated with secondary antibodies conjugated with the reporter enzyme HRP (DAKO, Denmark) prepared at 1:10000 in 0.05% PBS-Tween20 containing 5% of non-fat dried milk powder. For membrane developing were used the chemiluminescent HRP-substrate ECL (GE Healthcare) and membranes were exposed to a High-performance chemioluminescence film (Amersham Biosciences) in a dark room with a red light. Our stable isotope-labeled kinase assay-linked phosphoproteomics (siKALIP) MASTL experiments were based on the protocol previously described (28Xue L. Wang P. Cao P. Zhu J.-K. Tao W. Identification of ERK1 direct substrates using stable isotope labeled kinase assay-linked phosphoproteomics.Mol. Cell. Proteomics. 2014; 13: 3199-3210Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). First, 31×106 HEK293 6E of not transfected cells were lysed by sonication in 2 ml of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA), cell debris and insoluble particles were removed by centrifugation at 16,000 × g at 4 °C. The supernatant containing 400 μg of soluble protein was collected. The sample volume was adjusted to 200 μl with lysis buffer. To inhibit endogenous kinases in the lysate, the sample was incubated with 1 mm 5′-(4-fluorosulfonyl-benzoyl) adenosine (FSBA) with 10% DMSO for 1 h at 30 °C. After endogenous kinase inhibition, 10U FastAP phosphatase (ThermoFisher Scientific) and 23 μl of 10x rAPid phosphate buffer were supplemented to the sample and incubated for 3 h at 37 °C. Heat inactivation of the rAPid phosphatase was achieved by heating the sample for 5 mins at 75 °C. Excess of FSBA was removed by Vivacon filtration unit 30 kDa cut-off (Merck-Millipore). Concentrated samples were washed in the concentrator with 200 μl of lysis buffer and concentrated again. Samples in the filter were incubated in 300 μl of 1× kinase buffer (10 mm Tris pH 7.5, 50 mm KCl, 10 mm MgCl2, 1 mm DTT, 1 mm ATP(γ-P18O3) (Cambridge Isotope Laboratory, Andover, MA) and 100 nm of the desired MASTL construct (MASTL FL, Bonsai or 450) for 1 h at 30 °C. The reaction was quenched by Guanidine-HCl denaturation buffer (6 m Guanidine-HCl (GndCl), 100 mm Tris (pH 8.5), 5 mm Tris (2-carboxyethyl) phosphine and 10 mm chloroacetamide), to be further spun off the filter and heated for 10 mins at 99 °C. For i
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