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MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Supplementary Information Material and methods Chemicals The EGFR inhibitor NVP-AEE788 (Novartis), the Jak inhibitor I (Merck Calbiochem, #420099) and anisomycin (Alomone labs, # A-520) were prepared as 50 mM stock solutions in 100% DMSO. Doxorubicin (Adriablastin, Pfizer), EGF (Sigma Ref: E9644), PDGF (Sigma, Ref: P4306) and IL-4 (Sigma, Ref: I-4269) stock solutions were prepared as recommended by the manufacturer. For in vivo administration: Temodal (20 mg Temozolomide capsules, Essex Chemie AG, Luzern) was dissolved in 4 mL KZI/glucose (20/80, vol/vol); Taxotere was bought as 40 mg/mL solution (Sanofi Aventis, France), and prepared in KZI/glucose. Antibodies The primary antibodies used were as follows: anti-S473P-Akt (#9271), anti-T308P-Akt (#9276,), anti-S9P-GSK3β (#9336), anti-T389P-p70S6K (#9205), anti-YP/TP-Erk1/2 (#9101), anti-YP/TP-p38 (#9215), anti-YP/TP-JNK1/2 (#9101), anti-Y751P-PDGFR (#3161), anti- p21Cip1/Waf1 (#2946), anti-p27Kip1 (#2552) and anti-Ser15-p53 (#9284) antibodies were from Cell Signaling Technologies; anti-Akt (#05-591), anti-T32P-FKHRL1 (#06-952) and anti- PDGFR (#06-495) antibodies were from Upstate; anti-IGF-1R (#SC-713) and anti-EGFR (#SC-03) antibodies were from Santa Cruz; anti-GSK3α/β (#44610), anti-Y641P-Stat6 (#611566), anti-S1981P-ATM (#200-301), anti-T2609 DNA-PKcs (#GTX24194) and anti- 1 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Y1316P-IGF-1R were from Bio-Source International, Becton-Dickinson, Rockland, GenTex and internal production, respectively. The 4G10 antibody was from Millipore (#05-321MG). Cell lines The MCF7 (ATCC, #HTB-22), HT-29 (ATCC, #HTB-38), PC3M (MD Anderson Cancer Center, Houston, Texas, USA), and NCI-N87 (ATCC, #CRL-5822) cell lines were cultivated as described (3,4) and were authenticated by SNP analysis. CSF1R cell based assays A clone of HEK293 cells stably transfected with human CSF1R was used in a capture immunosorbant assay to measure autophosphorylation changes. One day before the assay 15000 cells/well were seeded into Poly-D-Lysine 96 well plates (Greiner Bio-One Cat. 655940). Serial dilutions in DMSO of test compounds were added in medium to cells for 90 min and then stimulated with CSF1R ligand MCSF (R&D Systems Cat. 216-MC) for 5-10 min. After removal of supernatant, cells were lyzed and lyzates transferred to a blocked MSD (MesoScale Discovery (MSD), Gaithersburg, MD, USA) plate that was pre-spotted with an antibody to CSF1R (SC-692, Santa Cruz Biotechnology) and incubated at 4oC overnight with shaking. The plates were washed and phosphorylation of captured CSF1R was detected with 10 nM SULFO-tag PY20, anti-phosphotyrosine detection antibody (MSD Cat. 32AP) added in 1% BSA/TBS/0.02% Tween-20. After shaking for one hour at room temperature, the plates were washed again and read on a Sector 6000 instrument (MSD) in 1.5 X Read T solution (MSD Cat. R93T). FGFR2 ELISA assay 2 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor The full description of the ELISA methodology has been recently described in Guagnano et al (2). In vivo angiogenic assays The chamber implant assay has been described previously (1). Tumor vascular permeability was assessed in rats bearing orthotopic BN472 tumors of approximately 2500 mm3 in size. Animals first received the Evans blue dye (0.5%, 5 ml/kg) via the femoral vein. Thirty minutes later, the high molecular weight vascular compartment marker FITC-dextran dye (Sigma, 100 mg/kg in PBS) was injected via the vena cava with the animals under isoflurane anaesthesia. Tumors were then excised 2 min later and snap frozen and sections prepared for the visualization and quantification of fluorescence as described in legend of Sup Figure 7B. 3 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Supplementary Table 1 Activity of NVP-BKM120 in the Invitrogen kinase panel Kinase Inh. (%) Kinase Inh. (%) Kinase Inh. (%) Kinase Inh. (%) ABL1 3 EPHB2 0 p38 alpha 4 PRKX 0 ABL1 E255K -4 EPHB3 -1 ERK1 7 PTK2 (FAK) 4 ABL1 G250E -4 EPHB4 -4 MAPKAPK2 -4 PTK6 (Brk) -10 ABL1 T315I 3 ErbB2 -4 MAPKAPK3 1 RET -4 ABL1 Y253F 1 EebB4 10 MAPKAPK5 -1 ROCK1 -2 ABL2 (Arg) -3 FER 1 HYL 0 ROCK2 3 GRK2 1 FES (FPS) 0 cMER 3 ROS1 27 GRK3 2 FGFR1 4 cMet 5 RSK1 5 Akt1 2 FGFR2 2 MET M1250T 1 RSK3 4 Akt2 0 FGFR3 2 MINK1 4 RSK2 2 Akt3 3 FGFR4 5 MST4 -3 MSK2 6 ALK 1 FGR 15 MUSK 5 MSK1 8 Aurora B 11 VEGFR1 2 skMLCK 8 p70S6K 8 BLK -3 FLT3 18 NEK1 8 SGK1 25 BMX 3 FLT3 D835Y 11 NEK2 7 SGK2 10 BTK 3 VEGFR3 2 TRKA 9 SGK3 20 CaMKI delta 12 PTK5 2 TRKB 9 SRC 10 CaMKII alpha 2 FYN 9 TRKC 4 SRC N1 7 CaMKII beta 9 GRK4 2 PAK3 -1 SRC N2 -1 CaMKII delta 1 GRK5 1 PAK4 -2 SRMS 13 CDK1/cyclin B 9 GRK6 4 PAK6 0 SRPK2 7 CDK2/cyclin A 2 GRK7 0 PASK -1 TSSK2 -6 CDK5/p35 -1 GSK3 alpha 5 PDGFRA 5 TSSK1 -4 CHK1 3 GSK3 beta 7 PDGFRA D842V 1 MST3 7 CHK2 17 HCK 10 PDGFRA T674I 11 YSK1 6 CLK1 32 IGF1R 1 PDGFRB 5 MST2 5 CLK2 15 IKK beta -2 PDK1 4 MST1 14 FMS (CSF1R) 85 INSR 6 PHKG1 11 Aurora A 34 CSK 5 INSRR (IRR) 3 PHKG2 3 SYK 1 CK1 alpha 1 -1 IRAK4 12 PIM1 6 TAOK2 6 CK1 delta 4 ITK 3 PIM2 3 TBK1 7 CK1 epsilon 11 JAK2 18 PKN1 (PRK1) 9 TEK 5 CK1 gamma 1 1 JAK3 7 PLK1 1 RSE 3 CK1 gamma 2 0 VEGFR2 3 PLK2 -2 YES1 12 CK1 gamma 3 -3 KIT 39 PLK3 1 CK2 alpha 1 1 KIT T670I 13 PKA 0 CK2 alpha 2 -2 LCK 7 PKC alpha 4 DAPK3 1 LYN A 6 PKC beta I 30 DYRK3 11 LYN B 3 PKC beta II 8 DYRK4 3 MEK1 4 PKC delta 4 EGFR 3 MEK2 3 PKC epsilon 2 EGFR L858R 12 MKK6 6 PKC gamma 5 EGFR L861Q 6 MLK1 2 PKC eta 4 EPHA1 -2 GCK 6 PKC iota 9 EPHA2 8 HGK 1 PKD3 8 EPHA3 -2 KHS1 10 PKC theta 5 EPHA4 9 ERK2 6 PKC zeta 2 EPHA5 0 p38 beta 4 PKC mu 5 EPHA8 3 p38 gamma 3 PKD2 2 EPHB1 3 p38 delta 2 PKG2 10 The data set is shown in full. No entries have been removed. 4 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Supplementary Table 2 Activity of NVP-BKM120 in the Ambit kinase panel Kinase Inh. (%) Kinase Inh. (%) Kinase Inh. (%) AAK1 0 CDC2L2 19 EGFR(S752-I759del) 0 ABL1 0 CDK11 0 EPHA1 2 ABL1(E255K) 0 CDK2 30 EPHA2 97.4 ABL1(F317I) 41 CDK3 0 EPHA3 0 ABL1(F317L) 0 CDK5 0 EPHA4 2 ABL1(H396P) 0 CDK7 0 EPHA5 0 ABL1(M351T) 0 CDK8 0 EPHA6 3 ABL1(Q252H) 0 CDK9 12 EPHA7 0 ABL1(T315I) 2 CDKL2 0 EPHA8 0 ABL1(Y253F) 0 CDKL3 0 EPHB1 0 ABL2 0 CDKL5 50 EPHB2 0 ACVR1 0 CHEK1 1 EPHB3 4 ACVR1B 3 CHEK2 0 EPHB4 4 ACVR2A 5 CIT 0 EPHB6 3 ACVR2B 0 CLK1 58 ERBB2 24 ACVRL1 38 CLK2 0 ERBB3 22 ADCK3 4 CLK3 29 ERBB4 7 ADCK4 0 CLK4 70 ERK1 1 AKT1 0 CSF1R 0 ERK2 0 AKT2 0 CSK 8 ERK3 0 AKT3 0 CSNK1A1L 0 ERK4 0 ALK 6 CSNK1D 27 ERK5 0 AMPK-alpha1 3 CSNK1E 7 ERK8 2 AMPK-alpha2 0 CSNK1G1 25 ERN1 5 ANKK1 0 CSNK1G2 0 FAK 21 ARK5 42 CSNK1G3 20 FER 0 ASK1 1 CSNK2A1 0 FES 0 ASK2 0 CSNK2A2 2 FGFR1 0 AURKA 0 CTK 28 FGFR2 98.4 AURKB 0 DAPK1 2 FGFR3 7 AURKC 0 DAPK2 13 FGFR3(G697C) 0 AXL 7 DAPK3 10 FGFR4 0 BIKE 0 DCAMKL1 7 FGR 0 BLK 0 DCAMKL2 30 FLT1 0 BMPR1A 0 DCAMKL3 51 FLT3 8 BMPR1B 0 DDR1 7 FLT3(D835H) 2 BMPR2 0 DDR2 19 FLT3(D835Y) 12 BMX 14 DLK 0 FLT3(ITD) 0 BRAF 18 DMPK 0 FLT3(K663Q) 0 BRAF(V600E) 17 DMPK2 0 FLT3(N841I) 6 BRK 0 DRAK1 0 FLT4 3 BRSK1 0 DRAK2 0 FRK 0 BRSK2 0 DYRK1A 11 FYN 0 BTK 6 DYRK1B 0 GAK 6 CAMK1 4 DYRK2 20 GCN2(Kin.Dom.2,S808G) 35 CAMK1D 0 EGFR 10 GRK1 0 CAMK1G 0 EGFR(E746-A750del) 0 GRK4 0 CAMK2A 2 EGFR(G719C) 4 GRK7 3 CAMK2B 18 EGFR(G719S) 10 GSK3A 42 CAMK2D 0 EGFR(L747-E749del, A750P 0 GSK3B 0 CAMK2G 0 EGFR(L747-S752del, P753S 1 HCK 0 CAMK4 50 EGFR(L747-T751del,Sins) 4 HIPK1 0 CAMKK1 0 EGFR(L858R) 16 HIPK2 0 CAMKK2 0 EGFR(L858R,T790M) 0 HIPK3 0 CDC2L1 10 EGFR(L861Q) 0 HIPK4 21 5 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Kinase Inh. (%) Kinase Inh. (%) Kinase Inh. (%) HPK1 0 MEK4 15 PHKG1 11 HUNK 18 MEK6 0 PHKG2 11 ICK 0 MELK 0 PIM1 2 IGF1R 0 MERTK 0 PIM2 0 IKK-alpha 29 MET 12 PIM3 5 IKK-beta 0 MET(M1250T) 20 PIP5K1A 0 IKK-epsilon 0 MET(Y1235D) 0 PIP5K2B 0 INSR 30 MINK 0 PKAC-alpha 0 INSRR 0 MKNK1 0 PKAC-beta 4 IRAK1 12 MKNK2 5 PKMYT1 0 IRAK3 0 MLCK 0 PKN1 0 ITK 0 MLK1 18 PKN2 0 JAK1(JH1) 38 MLK2 0 PLK1 30 JAK1(JH2) 0 MLK3 0 PLK2 0 JAK2(JH1) 12 MRCKA 0 PLK3 0 JAK3(JH1) 100 MRCKB 0 PLK4 1 JNK1 0 MST1 0 PRKCD 4 JNK2 0 MST1R 0 PRKCE 0 JNK3 17 MST2 0 PRKCH 0 KIT 0 MST3 5 PRKCQ 0 KIT(D816V) 3 MST4 0 PRKD1 36 KIT(L576P) 0 MUSK 0 PRKD2 38 KIT(V559D) 0 MYLK 12 PRKD3 11 KIT(V559D,T670I) 0 MYLK2 5 PRKG1 0 KIT(V559D,V654A 0MYO3A 7PRKG2 0 LATS1 4 MYO3B 1 PRKR 19 LATS2 19 NDR1 1 PRKX 0 LCK 0 NDR2 3 PRP4 4 LIMK1 0 NEK1 15 PYK2 0 LIMK2 0 NEK2 0 QSK 0 LKB1 0 NEK5 0 RAF1 7 LOK 4 NEK6 0 RET 8 LTK 0 NEK7 0 RET(M918T) 13 LYN 0 NEK9 0 RET(V804L) 12 LZK 0 NIM1 0 RET(V804M) 14 MAK 0 NLK 0 RIOK1 11 MAP3K1 0 OSR1 17 RIOK2 0 MAP3K15 0 p38-alpha 0 RIOK3 0 MAP3K2 0 p38-beta 0 RIPK1 0 MAP3K3 0 p38-delta 0 RIPK2 0 MAP3K4 1 p38-gamma 17 RIPK4 0 MAP4K2 17 PAK1 4 ROCK1 0 MAP4K3 0 PAK2 17 ROCK2 0 MAP4K4 9 PAK3 0 ROS1 0 MAP4K5 0 PAK4 13 RPS6KA1(Kin.Dom.1-N-term 0 MAPKAPK2 0 PAK6 16 RPS6KA1(Kin.Dom.2-C-term 23 MAPKAPK5 0 PAK7 0 RPS6KA2(Kin.Dom.1-N-term 2 MARK1 0 PCTK1 0 RPS6KA2(Kin.Dom.2-C-term 16 MARK2 0 PCTK2 26 RPS6KA3(Kin.Dom.1-N-term 0 MARK3 0 PCTK3 34 RPS6KA4(Kin.Dom.1-N-term 4 MARK4 0 PDGFRA 11 RPS6KA4(Kin.Dom.2-C-term 0 MAST1 0 PDGFRB 0 RPS6KA5(Kin.Dom.1-N-term 0 MEK1 0 PDPK1 0 RPS6KA5(Kin.Dom.2-C-term 50 MEK2 0 PFTAIRE2 0 RPS6KA6(Kin.Dom.1-N-term 0 MEK3 0 PFTK1 27 RPS6KA6(Kin.Dom.2-C-term 18 6 MCT-11-0474 BKM120: a potent and specific pan-PI3K inhibitor Kinase Inh.
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    Table S1. The numbers of KD genes in each KD time The number The number The number The number Cell lines of genes of genes of genes of genes (96h) (120h) (144h) PC3 3980 3822 128 1725 A549 3724 3724 0 0 MCF7 3688 3471 0 1837 HT29 3665 3665 0 0 A375 3826 3826 0 0 HA1E 3801 3801 0 0 VCAP 4134 34 4121 0 HCC515 3522 3522 0 0 Table S2. The predicted results in the PC3 cell line on the LINCS II data id target rank A07563059 ADRB2 48 A12896037 ADRA2C 91 A13021932 YES1 77 PPM1B;PPP1CC;PPP2CA; A13254067 584;1326;297;171;3335 PTPN1;PPP2R5A A16347691 GMNN 2219 PIK3CB;MTOR;PIK3CA;PIK A28467416 18;10;9;13;8 3CG;PIK3CD A28545468 EHMT2;MAOB 14;67 A29520968 HSPB1 1770 A48881734 EZH2 1596 A52922642 CACNA1C 201 A64553394 ADRB2 155 A65730376 DOT1L 3764 A82035391 JUN 378 A82156122 DPP4 771 HRH1;HTR2C;CHRM3;CH A82772293 2756;2354;2808;2367 RM1 A86248581 CDA 1785 A92800748 TEK 459 A93093700 LMNA 1399 K00152668 RARB 105 K01577834 ADORA2A 525 K01674964 HRH1;BLM 31;1314 K02314383 AR 132 K03194791 PDE4D 30 K03390685 MAP2K1 77 K06762493 GMNN;APEX1 1523;2360 K07106112 ERBB4;ERBB2;EGFR 497;60;23 K07310275 AKT1;MTOR;PIK3CA 13;12;1 K07753030 RGS4;BLM 3736;3080 K08109215 BRD2;BRD3;BRD4 1413;2786;3 K08248804 XIAP 88 K08586861 TBXA2R;MBNL1 297;3428 K08832567 GMNN;CA12 2544;50 LMNA;NFKB1;APEX1;EH K08976401 1322;341;3206;123 MT2 K09372874 IMPDH2 232 K09711437 PLA2G2A 59 K10859802 GPR119 214 K11267252 RET;ALK 395;760 K12609457 LMNA 907 K13094524 BRD4 7 K13662825 CDK4;CDK9;CDK5;CDK1 34;58;13;18 K14704277 LMNA;BLM 1697;1238 K14870255 AXL 1696 K15170068 MAN2B1 1756 K15179879
  • Kinase Profiling Book

    Kinase Profiling Book

    Custom and Pre-Selected Kinase Prof iling to f it your Budget and Needs! As of July 1, 2021 19.8653 mm 128 196 12 Tyrosine Serine/Threonine Lipid Kinases Kinases Kinases Carna Biosciences, Inc. 2007 Carna Biosciences, Inc. Profiling Assays available from Carna Biosciences, Inc. As of July 1, 2021 Page Kinase Name Assay Platform Page Kinase Name Assay Platform 4 ABL(ABL1) MSA 21 EGFR[T790M/C797S/L858R] MSA 4 ABL(ABL1)[E255K] MSA 21 EGFR[T790M/L858R] MSA 4 ABL(ABL1)[T315I] MSA 21 EPHA1 MSA 4 ACK(TNK2) MSA 21 EPHA2 MSA 4 AKT1 MSA 21 EPHA3 MSA 5 AKT2 MSA 22 EPHA4 MSA 5 AKT3 MSA 22 EPHA5 MSA 5 ALK MSA 22 EPHA6 MSA 5 ALK[C1156Y] MSA 22 EPHA7 MSA 5 ALK[F1174L] MSA 22 EPHA8 MSA 6 ALK[G1202R] MSA 23 EPHB1 MSA 6 ALK[G1269A] MSA 23 EPHB2 MSA 6 ALK[L1196M] MSA 23 EPHB3 MSA 6 ALK[R1275Q] MSA 23 EPHB4 MSA 6 ALK[T1151_L1152insT] MSA 23 Erk1(MAPK3) MSA 7 EML4-ALK MSA 24 Erk2(MAPK1) MSA 7 NPM1-ALK MSA 24 Erk5(MAPK7) MSA 7 AMPKα1/β1/γ1(PRKAA1/B1/G1) MSA 24 FAK(PTK2) MSA 7 AMPKα2/β1/γ1(PRKAA2/B1/G1) MSA 24 FER MSA 7 ARG(ABL2) MSA 24 FES MSA 8 AurA(AURKA) MSA 25 FGFR1 MSA 8 AurA(AURKA)/TPX2 MSA 25 FGFR1[V561M] MSA 8 AurB(AURKB)/INCENP MSA 25 FGFR2 MSA 8 AurC(AURKC) MSA 25 FGFR2[V564I] MSA 8 AXL MSA 25 FGFR3 MSA 9 BLK MSA 26 FGFR3[K650E] MSA 9 BMX MSA 26 FGFR3[K650M] MSA 9 BRK(PTK6) MSA 26 FGFR3[V555L] MSA 9 BRSK1 MSA 26 FGFR3[V555M] MSA 9 BRSK2 MSA 26 FGFR4 MSA 10 BTK MSA 27 FGFR4[N535K] MSA 10 BTK[C481S] MSA 27 FGFR4[V550E] MSA 10 BUB1/BUB3 MSA 27 FGFR4[V550L] MSA 10 CaMK1α(CAMK1) MSA 27 FGR MSA 10 CaMK1δ(CAMK1D) MSA 27 FLT1 MSA 11 CaMK2α(CAMK2A) MSA 28
  • Lipid Metabolic Reprogramming: Role in Melanoma Progression and Therapeutic Perspectives

    Lipid Metabolic Reprogramming: Role in Melanoma Progression and Therapeutic Perspectives

    cancers Review Lipid metabolic Reprogramming: Role in Melanoma Progression and Therapeutic Perspectives 1, 1, 1 2 1 Laurence Pellerin y, Lorry Carrié y , Carine Dufau , Laurence Nieto , Bruno Ségui , 1,3 1, , 1, , Thierry Levade , Joëlle Riond * z and Nathalie Andrieu-Abadie * z 1 Centre de Recherches en Cancérologie de Toulouse, Equipe Labellisée Fondation ARC, Université Fédérale de Toulouse Midi-Pyrénées, Université Toulouse III Paul-Sabatier, Inserm 1037, 2 avenue Hubert Curien, tgrCS 53717, 31037 Toulouse CEDEX 1, France; [email protected] (L.P.); [email protected] (L.C.); [email protected] (C.D.); [email protected] (B.S.); [email protected] (T.L.) 2 Institut de Pharmacologie et de Biologie Structurale, CNRS, Université Toulouse III Paul-Sabatier, UMR 5089, 205 Route de Narbonne, 31400 Toulouse, France; [email protected] 3 Laboratoire de Biochimie Métabolique, CHU Toulouse, 31059 Toulouse, France * Correspondence: [email protected] (J.R.); [email protected] (N.A.-A.); Tel.: +33-582-7416-20 (J.R.) These authors contributed equally to this work. y These authors jointly supervised this work. z Received: 15 September 2020; Accepted: 23 October 2020; Published: 27 October 2020 Simple Summary: Melanoma is a devastating skin cancer characterized by an impressive metabolic plasticity. Melanoma cells are able to adapt to the tumor microenvironment by using a variety of fuels that contribute to tumor growth and progression. In this review, the authors summarize the contribution of the lipid metabolic network in melanoma plasticity and aggressiveness, with a particular attention to specific lipid classes such as glycerophospholipids, sphingolipids, sterols and eicosanoids.
  • Article Reference

    Article Reference

    Article Phosphorylation by NLK inhibits YAP‐14‐3‐3‐interactions and induces its nuclear localization MOON, Sungho, et al. Reference MOON, Sungho, et al. Phosphorylation by NLK inhibits YAP‐14‐3‐3‐interactions and induces its nuclear localization. EMBO Reports, 2017, vol. 18, no. 1, p. 61-71 PMID : 27979972 DOI : 10.15252/embr.201642683 Available at: http://archive-ouverte.unige.ch/unige:112477 Disclaimer: layout of this document may differ from the published version. 1 / 1 Published online: December 15, 2016 Scientific Report Phosphorylation by NLK inhibits YAP-14-3-3- interactions and induces its nuclear localization Sungho Moon1,† , Wantae Kim1,†, Soyoung Kim1, Youngeun Kim1, Yonghee Song1, Oleksii Bilousov2, Jiyoung Kim1, Taebok Lee1, Boksik Cha1, Minseong Kim1, Hanjun Kim1, Vladimir L Katanaev2,3,* & Eek-hoon Jho1,** Abstract tissue homeostasis have become a long-standing topic of interest. As loss of the organ size control is linked to many human diseases, includ- Hippo signaling controls organ size by regulating cell proliferation ing cancer and degenerative diseases, regulation of the organ size and apoptosis. Yes-associated protein (YAP) is a key downstream could be an attractive therapeutic strategy. Recently, Hippo signaling effector of Hippo signaling, and LATS-mediated phosphorylation of has been identified as a major signaling pathway to control the organ YAP at Ser127 inhibits its nuclear localization and transcriptional size; dysregulation of this pathway results in aberrant growth [1]. activity. Here, we report that Nemo-like kinase (NLK) phosphory- The Hippo pathway is evolutionarily conserved from nematodes lates YAP at Ser128 both in vitro and in vivo, which blocks interac- to humans and controls a variety of cellular processes, such as cell tion with 14-3-3 and enhances its nuclear localization.
  • Phosphorylation of Threonine 3 on Histone H3 by Haspin Kinase Is Required for Meiosis I in Mouse Oocytes

    Phosphorylation of Threonine 3 on Histone H3 by Haspin Kinase Is Required for Meiosis I in Mouse Oocytes

    ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 5066–5078 doi:10.1242/jcs.158840 RESEARCH ARTICLE Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes Alexandra L. Nguyen, Amanda S. Gentilello, Ahmed Z. Balboula*, Vibha Shrivastava, Jacob Ohring and Karen Schindler` ABSTRACT a lesser extent. However, it is not known whether haspin is required for meiosis in oocytes. To date, the only known haspin substrates Meiosis I (MI), the division that generates haploids, is prone to are threonine 3 of histone H3 (H3T3), serine 137 of macroH2A and errors that lead to aneuploidy in females. Haspin is a kinase that threonine 57 of CENP-T (Maiolica et al., 2014). Knockdown or phosphorylates histone H3 on threonine 3, thereby recruiting Aurora inhibition of haspin in mitotically dividing tissue culture cell kinase B (AURKB) and the chromosomal passenger complex lines reveal that phosphorylation of H3T3 is essential for the (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an alignment of chromosomes at the metaphase plate (Dai and AURKB homolog, are enriched in germ cells, yet their significance Higgins, 2005; Dai et al., 2005), regulation of chromosome in regulating MI is not fully understood. Using inhibitors and cohesion (Dai et al., 2009) and establishing a bipolar spindle (Dai overexpression approaches, we show a role for haspin during MI et al., 2009). In mitotic metaphase, phosphorylation of H3T3 is in mouse oocytes. Haspin-perturbed oocytes display abnormalities restricted to kinetochores, and this mark signals recruitment of the in chromosome morphology and alignment, improper kinetochore– chromosomal passenger complex (CPC) (Dai et al., 2005; Wang microtubule attachments at metaphase I and aneuploidy at et al., 2010; Yamagishi et al., 2010).