A subcellular map of the human kinome

  1. Haitao Zhang
  2. Xiaolei Cao
  3. Mei Tang
  4. Guoxuan Zhong
  5. Yuan Si
  6. Haidong Li
  7. Feifeng Zhu
  8. Qinghua Liao
  9. Liuju Li
  10. Jianhui Zhao
  11. Jia Feng
  12. Shuaifeng Li
  13. Chenliang Wang
  14. Manuel Kaulich
  15. Fangwei Wang
  16. Liangyi Chen
  17. Li Li
  18. Zongping Xia
  19. Tingbo Liang
  20. Huasong Lu
  21. Xin-Hua Feng
  22. Bin Zhao  Is a corresponding author
  1. The MOE Key Laboratory of Biosystems Homeostasis & Protection, Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, China
  2. Cancer Center, Zhejiang University, China
  3. College of Biology and Pharmacy, Yulin Normal University, China
  4. State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, China
  5. Department of Hepatobiliary and Pancreatic Surgery, Zhejiang Provincial Key Laboratory of Pancreatic Disease, The First Affiliated Hospital, School of Medicine, Zhejiang University, China
  6. Department of Ophthalmology, The Children’s Hospital, School of Medicine, and National Clinical Research Center for Child Health, Zhejiang University, China
  7. Institute of Biochemistry II, Goethe University Frankfurt-Medical Faculty, University Hospital, Germany
  8. Institute of Aging Research, Hangzhou Normal University, China
9 figures, 1 table and 7 additional files

Figures

Figure 1 with 1 supplement
Mapping subcellular localizations of the kinome.

(A) Coverage of the kinome (pie graph) and kinase families (bar graph) by the plasmid library. (B) Composition of the kinome library. Species origins (left), epitope tags (middle), and tag positions …

Figure 1—figure supplement 1
Mapping subcellular localizations of the kinome.

(A) Distribution of the kinome plasmid library on the kinome tree. Each red dot represents a kinase in the library. (B) Ratio of N-terminal- and C-terminal-tagged kinases in each kinase family. (C) …

Figure 2 with 1 supplement
Kinome Atlas (KA) complements current databases for kinome localization.

(A) Coverage of the kinome by COPARTMENTS, Human Protein Atlas (HPA), and KA in Venn diagram. (B) Subcellular localization of six kinases first annotated by KA. (C, D) Comparison of annotations in …

Figure 2—figure supplement 1
Kinome Atlas (KA) complements current databases for kinome localization.

(A) Comparison of specific localizations in KA with high-confidence localizations in COMPARTMENTS. Distributions of matched and unmatched localizations are shown by bar graph. (B) The heatmap …

Figure 3 with 1 supplement
Kinome Atlas (KA) identified unknown localizations for kinases.

(A, B) New plasma membrane-localized kinases identified by KA. Original mapping data (A) and confirmation by cellular fractionation (B). W: whole cell lysate; M: membrane; C: cytosol. Equal loading …

Figure 3—figure supplement 1
Kinome Atlas (KA) identified unknown localizations for kinases.

(A) C-terminal tagged SGK3 colocalizes with EEA1 in HeLa cells. (B) PRKD3, STK16, MAP3K12, and MAP3K13 are not secreted. HEK293T cells were transfected. Both culture medium and cell lysates were …

Figure 4 with 1 supplement
Phase separation assembles kinase condensates.

(A) Sub-nuclear patterns of kinases in Kinome Atlas (KA). (B) Hex disrupts puncta localization of kinases. Transfected HeLa cells were treated with 10% 1,6-hexanediol (Hex) for 1 min as indicated. …

Figure 4—figure supplement 1
Phase separation assembles kinase condensates.

(A) Nuclear peripheral kinases visualized by Airyscan high-resolution microscopy. Transfected HeLa cells were stained with anti-HA for the kinase and anti-Lamin A/C for nuclear envelope. (B) …

Figure 5 with 1 supplement
Kinome Atlas (KA) identified novel mitochondrial kinases.

(A) Comparison of MitoCarta2.0 and KA. (B) FASTK and PAK5 are not mitochondrial. N- and C-terminal tagged kinases were expressed in HeLa cells and stained. (C) Mitochondrial kinases identified by …

Figure 5—figure supplement 1
Kinome Atlas (KA) identified novel mitochondrial kinases.

(A) Confirmation of mitochondrial kinases by co-staining with TOM20. (B) N-terminal truncated isoform of FASTK exhibited mitochondrial localization. FASTK-M35 was transfected in HeLa cells and …

Figure 6 with 1 supplement
MOK is a mitochondrial intermembrane space (IMS)-localized protein.

(A) Sub-mitochondrial localizations of new mitochondrial kinases. Transfected HeLa cells were stained and imaged by 3D structured illumination microscopy (3D-SIM). (B) MOK2 is imported into …

Figure 6—figure supplement 1
MOK is a mitochondrial intermembrane space (IMS)-localized protein.

(A) Domain organization of LIMK2 isoforms. (B) Subcellular localization of LIMK2 isoforms. HeLa cells transfected with LIMK2 isoforms were stained with anti-Flag and anti-TOM20 antibodies. F-actin …

Figure 7 with 1 supplement
Knockout (KO) of MOK impairs mitochondrial structure and functions.

(A, B) MOK KO reduced mitochondria cristae. Indicated A375 cells were examined by electron microscopy (A). The length and cristae number of mitochondria (n = 30) were quantified (B). (C, D) MOK KO …

Figure 7—figure supplement 1
Knockout of MOK impairs mitochondrial structure and functions.

(A) Confirmation of MOK knockout in A375 cells by genomic DNA sequencing. Indels are labeled in red. Arrowhead indicates predicted Cas9 cutting site. (B) MOK knockdown efficiency in Caki-1 cells as …

Author response image 1
Expression of MOK in human and mouse tissues.

Data was analyzed from the HPA RNA-seq normal tissues project (human) and the Mouse ENCODE transcriptome data.

Author response image 2
Expression of MOK in human cancers in TCGA database as analyzed by FireBrowse (http://firebrowse.org/).

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)Human kinome cDNACenter for Cancer Systems Biology of Harvard UniversityhORFeome v3.1
Gene (Homo sapiens)Human kinome cDNAThermo FisherUltimate ORF Clone LITE Collection
Cell line (Homo sapiens)HeLaATCCCat# CCL-2, RRID:CVCL_0030
Cell line (Homo sapiens)HEK293TATCCCat# CRL-11268, RRID:CVCL_1926
Cell line (Homo sapiens)A375ATCCCat# CRL-1619, RRID:CVCL_0132
Cell line (Homo sapiens)DU 145ATCCCat# HTB-81, RRID:CVCL_0105
Cell line (Homo sapiens)MCF10AATCCCat# CRL-10317, RRID:CVCL_0598
Cell line (Homo sapiens)Caki-1Liping Xie (Zhejiang University)
Cell line (Cercopithecus aethiops)COS-7ATCCCat# CRL-1651, RRID:CVCL_0224
Transfected construct (Homo sapiens)siNTThis paperControl siRNA target sequence: UUCUCCGAACGUGUCA
CGU
Transfected construct (Homo sapiens)siMOK#1This papersiRNA target sequence: CCUCUUUCCUGGAGUA
AAU
Transfected construct (Homo sapiens)siMOK#2This papersiRNA target sequence: AGUCGAGAGCUAUGAA
UUU
Transfected construct (Homo sapiens)shNTThis paperControl shRNA target sequence: CCTAAGGTTAAGTCGCCCTCG
Transfected construct (Homo sapiens)sh-TOM20This papershRNA target sequence: GGCGTAGACCATCTGACAAAT
Transfected construct (Homo sapiens)sh-TOM70This papershRNA target sequence:
GCATGCTGTTAGCCGATAAAG
Transfected construct (Homo sapiens)sh-TIM17AThis papershRNA target sequence:
GCTGGTATCTTGTTGACAAGA
Transfected construct (Homo sapiens)sh-TIM50This papershRNA target sequence:
CCTCAAGACCATTGCACTGAA
Transfected construct (Homo sapiens)sh-TIM23This papershRNA target sequence:
CCAGCCTCTATGCACTATATA
Transfected construct (Homo sapiens)sh-HSPA9This papershRNA target sequence:
CGTGCTCAATTTGAAGGGATT
Transfected construct (Homo sapiens)sh-HSP60This papershRNA target sequence:
CCTGCTCTTGAAATTGCCAAT
Transfected construct (Homo sapiens)sh-MIA40This papershRNA target sequence:
TCTTGACATCTTGACATATAC
Transfected construct (Homo sapiens)sh-TIM9This papershRNA target sequence:
GCTTGGTCACTTGATTAGAAA
Transfected construct (Homo sapiens)sh-TIM22This papershRNA target sequence:
GCTTTGACCCTAAGGATCCTT
Transfected construct (Homo sapiens)sh-OXA1LThis papershRNA target sequence:
CGAATCAGAGAGGCCAAGTTA
Transfected construct (Homo sapiens)sh-BCS1LThis papershRNA target sequence:
CGTCCAGGAATTCATCGATAA
Transfected construct (Homo sapiens)sh-MOK #1This papershRNA target sequence:
CTGGTTCTCTTGCAC TAATAT
Transfected construct (Homo sapiens)sh-MOK #2This papershRNA target sequence:
ACCTCTACTAACAACCAATTT
Transfected construct (Homo sapiens)sh-ATAD3A #1This papershRNA target sequence:
CATCAATGAGATGGTCCACTT
Transfected construct (Homo sapiens)sh-ATAD3A #2This papershRNA target sequence:
CAAGGACAAATGGAGCAACTT
Transfected construct (Homo sapiens)sg-MOK KO #1This paperPEP-KO with gRNA sequence: GTACCAGTTATGTAAGTCCC
Transfected construct (Homo sapiens)sg-MOK KO #2This paperPEP-KO with gRNA sequence: GCAATTGGCAAAATAGGAGA
Transfected construct (Homo sapiens)sg-BMP2K KI #1This paperPEP-KI with gRNA sequence: GAAATGGAGCAGCACCAAAT
Transfected construct (Homo sapiens)sg-BMP2K KI #2This paperPEP-KI with gRNA sequence: TAAACAGTAGATACTTCTGA
Transfected construct (Homo sapiens)sg-MOK KI #1This paperPEP-KI with gRNA sequence: TCTTCCGCCTTTCCGCACTA
Transfected construct (Homo sapiens)sg-MOK KI #2This paperPEP-KI with gRNA sequence: CACCGTCGTCTCGACTTCGG
AntibodyMouse monoclonal anti-alpha 1 Sodium Potassium ATPase antibodyAbcamCat# ab7671, RRID:AB_306023WB (1:2000)
AntibodyMouse monoclonal anti-GM130 antibodyBD BiosciencesCat# 610823, RRID:AB_398142IF (1:1000)
AntibodyRabbit polyclonal anti-UTP25 antibodyDr. Jinrong Peng (Zhejiang University)IF (1:200)
AntibodyRabbit polyclonal anti-Lamin A/C (H-110) antibodySanta Cruz BiotechnologyCat# sc-20681, RRID:AB_648154WB (1:2000)
AntibodyMouse monoclonal anti-Lamin A/C (636) antibodySanta Cruz BiotechnologyCat# sc-7292, RRID:AB_627875IF (1:1000)
AntibodyRabbit monoclonal anti-Catalase (D4P7B) antibodyCell Signaling TechnologyCat# 12980, RRID:AB_2798079IF (1:100)
AntibodyRabbit polyclonal anti-Pericentrin antibodyCovanceCat# PRB-432C-200, RRID:AB_291635IF (1:500)
AntibodyRabbit monoclonal anti-EEA1 (C45B10) antibodyCell Signaling TechnologyCat# 3288, RRID:AB_2096811IF (1:100)
AntibodyRabbit monoclonal anti-TOM20 (F-10) antibodySanta Cruz BiotechnologyCat# sc-17764, RRID:AB_628381IF (1:1000)
AntibodyMouse polyclonal anti-TOM20 (FL-145) antibodySanta Cruz BiotechnologyCat# sc-11415, RRID:AB_2207533IF (1:1000)
AntibodyRabbit monoclonal anti-HSP60 (D6F1) antibodyCell Signaling TechnologyCat# 46611, RRID:AB_2799305WB (1:1000),
IF (1:500)
AntibodyMouse monoclonal anti-ENDOG (B-2) antibodySanta Cruz BiotechnologyCat# sc-365359, RRID:AB_10843802WB (1:2000)
AntibodyMouse monoclonal anti-MT-CO2 antibodyAbcamCat# ab110258, RRID:AB_10887758WB (1:2000)
AntibodyMouse monoclonal anti-LAMP1 antibodySanta Cruz BiotechnologyCat# sc-20011, RRID:AB_626853IF (1:500)
AntibodyMouse monoclonal anti-HSP90 antibodyBD BiosciencesCat# 610418, RRID:AB_397798WB (1:5000)
AntibodyRabbit monoclonal anti-pErk1/2 (Thr202/Tyr204) antibodyCell Signaling TechnologyCat# 4370, RRID:AB_2315112WB (1:2000)
AntibodyRabbit monoclonal anti-Thiophosphate ester antibodyAbcamCat# ab133473, RRID:AB_2737094WB (1:5000)
AntibodyRabbit monoclonal anti-PARP (46D11) antibodyCell Signaling TechnologyCat# 9532, RRID:AB_659884WB (1:2000)
AntibodyRabbit polyclonal anti-RPS6KA6 antibodySigma-AldrichCat# HPA002852, RRID:AB_1079854IF (1:100)
AntibodyRabbit polyclonal anti-MOK antibodySigma-AldrichCat# HPA027292, RRID:AB_10600989WB (1:1000)
AntibodyMouse monoclonal ANTI-FLAG M2 antibodySigma-AldrichCat# F3165, RRID:AB_259529IF (1:1000)
AntibodyRabbit monoclonal anti-DYKDDDDK Tag (D6W5B) antibodyCell Signaling TechnologyCat# 14793, RRID:AB_2572291IF (1:500)
AntibodyMouse monoclonal anti-Flag M2-Peroxidase-HRP conjugated antibodySigma-AldrichCat# A8592, RRID:AB_439702WB (1:5000)
AntibodyRabbit monoclonal anti-HA-Tag (C29F4) antibodyCell Signaling TechnologyCat# 3724, RRID:AB_1549585IF (1:500)
AntibodyMouse monoclonal anti-Myc-Tag (9B11) antibodyCell Signaling TechnologyCat# 2276, RRID:AB_331783IF (1:500)
AntibodyRabbit polyclonal anti-GFP antibodyAbcamCat# ab6556, RRID:AB_305564WB (1:2000)
AntibodyMouse monoclonal anti-β-actin antibodyProteintechCat# 66009–1-Ig, RRID:AB_2687938WB (1:5000)
AntibodyMouse monoclonal anti-Tubulin (clone B5-1-2) antibodySigma-AldrichCat# T6074, RRID:AB_477582WB (1:5000),
IF (1:1000)
AntibodyMouse monoclonal anti- γ-Tubulin (clone GTU88) antibodySigma-AldrichCat# T5326, RRID:AB_532292IF (1:500)
AntibodyGoat polyclonal anti-Rabbit IgG(H + L)-Alexa Fluor 488 conjugated antibodyThermo Fisher ScientificCat# A-11034, RRID:AB_2576217IF (1:1000)
AntibodyGoat polyclonal anti-Mouse IgG(H + L)-Alexa Fluor 488 conjugated antibodyThermo Fisher ScientificCat# A-11029, RRID:AB_2534088IF (1:1000)
AntibodyGoat polyclonal anti-Rabbit IgG(H + L)-Alexa Fluor 594 conjugated antibodyThermo Fisher ScientificCat# A-11037, RRID:AB_2534095IF (1:1000)
AntibodyGoat polyclonal anti-Mouse IgG(H + L)-Alexa Fluor 594 conjugated antibodyThermo Fisher ScientificCat# A-11032, RRID:AB_2534091IF (1:1000)
AntibodyGoat polyclonal anti-Rabbit IgG(H + L)-Alexa Fluor 647 conjugated antibodyThermo Fisher ScientificCat# A-21244, RRID:AB_2535812IF (1:1000)
Sequence-based reagentTOM20 RT-FThis paperAGGGCGTAGACCATCTGACA
Sequence-based reagentTOM20 RT-RThis paperTTCAGCCAAGCTCTGAGCAC
Sequence-based reagentTOM70 RT-FThis paperAGACGTGCAAAAGCCCATGA
Sequence-based reagentTOM70 RT-RThis paperAAGCATGGGCTGGGAAATGA
Sequence-based reagentTIM17A RT-FThis paperGGGAGGGCTGTTTTCCATGA
Sequence-based reagentTIM17A RT-RThis paperGGAGGGGTCTTCTGCAAACT
Sequence-based reagentTIM50 RT-FThis paperTGCACGAGGTTGGCGA
Sequence-based reagentTIM50 RT-RThis paperTGTATGTCCGGCGCAACTG
Sequence-based reagentTIM23 RT-FThis paperAGGGGCACTTTGGGCTAATAC
Sequence-based reagentTIM23 RT-RThis paperTATCCCTCGAAGACCACCTGT
Sequence-based reagentHSPA9 RT-FThis paperCCTACGGCCTGGACAAGAAG
Sequence-based reagentHSPA9 RT-RThis paperCTTGTGCTTGCGCTTGAACT
Sequence-based reagentHSP60 RT-FThis paperCTTTTAGCCGATGCTGTGGC
Sequence-based reagentHSP60 RT-RThis paperTTGGCTATAGAGCGTGCCAG
Sequence-based reagentMIA40 RT-FThis paperCTATTGCCGGCAGGAAGGG
Sequence-based reagentMIA40 RT-RThis paperGGCATGGGCAGTTCCAGTTA
Sequence-based reagentTIM9 RT-FThis paperACAGAGACCTGCTTTTTGGACT
Sequence-based reagentTIM9 RT-RThis paperGCCAGGGCTTCATTCTGCTG
Sequence-based reagentTIM21 RT-FThis paperCCTGAGACAGCGGGTTCC
Sequence-based reagentTIM21 RT-RThis paperAAGACAAATCCTCCCACGCA
Sequence-based reagentOXA1L RT-FThis paperCTCGCAATGGCTTGGGAAAC
Sequence-based reagentOXA1L RT-RThis paperCTCAGGTACTGCTGTGGGTG
Sequence-based reagentBCS1L RT-FThis paperATGGCGTCCCTTTGGCTATC
Sequence-based reagentBCS1L RT-RThis paperAGTCGGTCATCAGAGAGGCT
Sequence-based reagentMOK RT-FThis paperTGAGCTAATACGAGGGAGAAGA
Sequence-based reagentMOK RT-RThis paperTACTCCAGGAAAGAGGGGCT
Sequence-based reagentATAD3A RT-FThis paperGCCTCCTGCTCTTTGTGGAT
Sequence-based reagentATAD3A RT-RThis paperAACTGCTCTGGTTGGTTGCT
Sequence-based reagentACTB RT-FThis paperCTCGCCTTTGCCGATCC
Sequence-based reagentACTB RT-RThis paperGAATCCTTCTGACCCATGCC
Commercial assay or kitSeahorse XF Cell Mito Stress Test KitAgilent TechnologiesCat# 103015-100
Commercial assay or kitATP detection kitBeyotime BiotechnologyCat# S0026
Chemical compound, drugProLong Gold Antifade Mountant with DAPIThermo Fisher ScientificCat# P36931
Chemical compound, drugAlexa Fluor 488 phalloidinThermo Fisher ScientificCat# A12379IF (1:1000)
Chemical compound, drugMitoTracker Green FMThermo Fisher ScientificCat# M7514
Chemical compound, drugCM-H2DCFDAThermo Fisher ScientificCat# C6827
Chemical compound, drugMitoTracker Red CMXRosThermo Fisher ScientificCat# M7512
Chemical compound, drugPfu Turbo DNA PolymeraseAgilent TechnologiesCat# 600252-52
Chemical compound, drugStreptavidin ResinAgilent TechnologiesCat# 240105--51
Chemical compound, drug1,6-HexanediolSigma-AldrichCat# 8043081000
Chemical compound, drugEDTA-free Protease Inhibitor CocktailSigma-AldrichCat# S8830
Chemical compound, drugANTI-FLAG M2 agarose beadsSigma-AldrichCat# F2426
Chemical compound, drugPolybreneSigma-AldrichCat# 107689
Chemical compound, drugDiamideSigma-AldrichCat# D3648
Chemical compound, drugBiotinSigma-AldrichCat# B4501
Chemical compound, drugVacuolin-1Sigma-AldrichCat# V7139
Chemical compound, drugDigitoninSangon BiotechCat# DG1152
Chemical compound, drugATP-γ-SAbcamCat# ab138911
Chemical compound, drugp-Nitrobenzyl mesylate (PNBM)AbcamCat# ab138910
Software, algorithmGraphPad PrismGraphPadRRID:SCR_002798
Software, algorithmImage-Pro Plus 6.0Image-Pro PlusRRID:SCR_016879
Software, algorithmVENNY2.1https://bioinfogp.cnb.csic.es/tools/venny/index.htmlCoverage of the kinome by KA and databases were analyzed by VENNY2.1
Software, algorithmKinMapPMID:28056780Kinome Atlas were presented on kinome tree using KinMap The localization and enrichment levels were reflected by colors and sizes of circles
Software, algorithmSMARThttp://smart.embl.de/Domain architecture of each kinase is analyzed by SMART
Software, algorithmIUPred2Ahttps://iupred2a.elte.hu/Intrinsic disorder tendency is predicted by IUPred2A
Software, algorithmPLAAChttp://plaac.wi.mit.edu/Prion-like domains (PrD.like, red) of each kinase are predicted by PLAAC

Additional files

Supplementary file 1

Human kinome and the kinome plasmid library, related to Figure 1A, B, Figure 1—figure supplement 1A, B, and Figure 2A.

https://cdn.elifesciences.org/articles/64943/elife-64943-supp1-v2.xlsx
Supplementary file 2

Summary of the Kinome Atlas, related to Figure 1E–G, Figure 1—figure supplement 1D, and Figure 2—figure supplement 1C.

https://cdn.elifesciences.org/articles/64943/elife-64943-supp2-v2.xlsx
Supplementary file 3

Coverage of the kinome by Kinome Atlas (KA) and databases, related to Figure 2A.

https://cdn.elifesciences.org/articles/64943/elife-64943-supp3-v2.xlsx
Supplementary file 4

Kinome Atlas (KA) in comparison with COMPARTMENTS, related to Figure 2C, D.

https://cdn.elifesciences.org/articles/64943/elife-64943-supp4-v2.xlsx
Supplementary file 5

Kinome Atlas (KA) in comparison with Human Protein Atlas (HPA), related to Figure 2E, F, Figure 2—figure supplement 1D, E.

https://cdn.elifesciences.org/articles/64943/elife-64943-supp5-v2.xlsx
Supplementary file 6

Analysis of kinases uniquely annotated by Kinome Atlas (KA), related to Figure 2G,H and Figure 3.

https://cdn.elifesciences.org/articles/64943/elife-64943-supp6-v2.xlsx
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