National Heart, Lung, and Blood Institute

Epithelial Systems Biology Laboratory (ESBL)


CRISPR Knockout Screening to Identify Protein Kinases That Affect Vasopressin-Stimulated Aqp2 Gene Transcription

This database lists the “hits” from a kinome-wide CRISPR knockout screen to identify protein kinases that regulate vasopressin-dependent Aqp2 gene transcription in a polarized mouse renal collecting duct cell line (mpkCCD). The screen utilized Cas9-expressing mpkCCD cells featuring a GFP-reporter inserted at the 5’ end of the Aqp2 gene allowing it to be co-transcribed with Aqp2. The cells were grown on permeable supports and treated with a vasopressin V2 receptor-selective analog (dDAVP). A lentiviral kinome library, targeting 713 kinase and kinase-related genes (n=4 guide RNAs per target), was introduced at low multiplicity of infection (MOI ≈0.1). The cells were sorted into populations based on their GFP fluorescence intensity using FACS. The genomic DNA was then extracted, and guide RNA counts were derived for each faction using Next-Generation Sequencing (NGS). Computational analysis using MAGeCK (https://sourceforge.net/p/mageck/wiki/Home/) identified kinases whose deletion significantly enriched (negative regulators) or depleted (positive regulators) particular guide RNAs in the high- versus low-GFP populations.

Database was created by Euijung Park, Lihe Chen, and Mark Knepper in the Epithelial Systems Biology Laboratory (Mark Knepper, Chief) at the National Heart, Lung and Blood Institute as part of its Kidney Systems Biology Project. Reference: Pending. Click to download processed data as spreadsheet.

Kinome CRISPR Screen Hits:

Gene Symbol Annotation Category Kinase Effect on Aqp2 Transcription General Reference*
Prkaca cAMP-activated kinase, PKA catalytic subunit a Vasopressin signaling network positive regulation Taylor et al., 2004. PMID: 15023366.
Prkar1a cAMP-activated kinase inhibitor, PKA regulatory subunit, type Ia Vasopressin signaling network negative regulation Taylor et al., 2004. PMID: 15023366.
Akt1 Basophilic kinase, Protein Kinase B (PKB) Vasopressin signaling network positive regulation Du &Montminy M, 1998, PMID 9829964
Stk11 Catalytic subunit of liver kinase B 1 (LKB1) complex Vasopressin signaling network negative regulation Alessi et al., 2006, PMID: 16756488.
Strada LKB1 subunit (required for LKB1 activity) Vasopressin signaling network negative regulation Alessi et al., 2006, PMID: 16756488.
Mark2 Par1b, a key regulator of cell polarity Vasopressin signaling network positive regulation Suzuki et al., 2004, PMID: 15324659.
Gsk3b Multifunctional regulator Vasopressin signaling network positive regulation Force &Woodgett, 2009, PMID: 19064989.
Tgfbr1 Epithelial-to-mesenchymal transition negative regulation Massagué, 1998 , PMID: 9759503.
Tgfbr2 Subunit of TGF-β receptor complex Epithelial-to-mesenchymal transition negative regulation Massagué, 1998 , PMID: 9759503.
Tgfbr3 Accessory protein of  TGF-β receptor complex Epithelial-to-mesenchymal transition negative regulation Massagué, 1998 , PMID: 9759503.
Dyrk1a Activator of DREAM complex assembly/quiescence Cell cycle regulation network positive regulation Litovchick et al., 2011, PMID: 21498570.
Gsk3b Multifunctional regulator Cell cycle regulation network positive regulation Force &Woodgett, 2009, PMID: 19064989.
Cdk10 Cyclin M dependent cell cycle regulator Cell cycle regulation network positive regulation Li et al., 1995, PMID: 7664269.
Nek7 A cell-cycle regulation kinase Cell cycle regulation network positive regulation Belham C, 2001, PMID: 11516946.
Cdk12 Positive regulator of transcriptional elongation Regulation of transcriptional apparatus positive regulation Bowman& Kelly, 2014, PMID: 24879308.
Tlk2 Stabilizes chromatin assembly factor ASF1A  Regulation of transcriptional apparatus positive regulation Silljé & Nigg,  EA., 2001, PMID: 11470414.
Map2k7 Element of  c-Jun N-terminal Kinase (JNK) cascade MAPK Pathways negative regulation Zou et al., 2006, PMID: 17187786
Map2k4 Element of  c-Jun N-terminal Kinase (JNK) cascade MAPK Pathways negative regulation Zou et al., 2006, PMID: 17187787
Map3k1 Element of  c-Jun N-terminal Kinase (JNK) cascade MAPK Pathways negative regulation Fuchs et al., 1998,  PMID: 9724739.
Map2k3 Element of p38 cascade MAPK Pathways positive regulation Dérijard et al., 1995, PMID: 7839144.
Wnk4 Regulator of ion transport in kidney epithelia  Ion transport regulation negative regulation Huang et al., 2007, PMID: 18059132.
Oxsr1 Activated by Wnk4 mediated phosphorylation Ion transport regulation negative regulation Chen et al., 2004, PMID: 14707132.
Ulk1 Activator of aldosterone receptor through phosphorylation Ion transport regulation negative regulation Shibata et al., 2018, PMID: 30021155.
Pim3 Aldosterone-induced Inhibitor of apoptosis Ion transport regulation positive regulation Mukaida et al., 2011, PMID: 21518143.
Csnk1a1 Casein kinase I isoform a Wnt pathway positive regulation Liu et al., 2002, PMID: 11955436.
Gsk3b Multifunctional regulator Wnt pathway positive regulation Force &Woodgett, 2009, PMID: 19064989.
Stk40 Tribbles-like pseudoknase Pseudokinases positive regulation Durzynska et al, 2017, PMID: 28089446.
Trib1 Member of tribbles pseudoknase family Pseudokinases positive regulation Kiss-Toth, 2011, PMID: 21428962.
* Full References:
1. Taylor SS, Yang J, Wu J, Haste NM, Radzio-Andzelm E, Anand G. PKA: a portrait of protein kinase dynamics. Biochim Biophys Acta. 2004 Mar 11;1697(1-2):259-69. 
2. Du K, Montminy M (1998). CREB Is a Regulatory Target for the Protein Kinase Akt/PKB. J BiolChem. 273 (49): 32377–32379. 
3. Alessi DR, Sakamoto K, Bayascas JR. LKB1-dependent signaling pathways. Annu Rev Biochem. 2006;75:137-63. 
4. Suzuki A, Hirata M, Kamimura K, Maniwa R, Yamanaka T, Mizuno K, Kishikawa M, Hirose H, Amano Y, Izumi N, Miwa Y, Ohno S. aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity. Curr Biol. 2004 Aug 24;14(16):1425-35. 
5. Force T, Woodgett JR. Unique and overlapping functions of GSK-3 isoforms in cell differentiation and proliferation and cardiovascular development. J Biol Chem. 2009 ;284: 9643-7. 
6. Massagué J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753-91. 
7. Litovchick L, Florens LA, Swanson SK, Washburn MP, DeCaprio JA. DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly. Genes Dev. 2011 Apr 15;25(8):801-13. 
8. Li S, MacLachlan TK, De Luca A, Claudio PP, Condorelli G, Giordano A. The cdc-2-related kinase, PISSLRE, is essential for cell growth and acts in G2 phase of the cell cycle. Cancer Res. 1995 Sep 15;55(18):3992-5. 
9. Belham C, Comb MJ, Avruch J. Identification of the NIMA family kinases NEK6/7 as regulators of the p70 ribosomal S6 kinase. Curr Biol. 2001 Aug 7;11(15):1155-67. 
10. Bowman EA, Kelly WG. RNA polymerase II transcription elongation and Pol II CTD Ser2 phosphorylation: A tail of two kinases. Nucleus. 2014 May-Jun;5(3):224-36. 
11. Silljé HH, Nigg EA. Identification of human Asf1 chromatin assembly factors as substrates of Tousled-like kinases. Curr Biol. 2001 Jul 10;11(13):1068-73. 
12. Zou H, Li Q, Lin SC, Wu Z, Han J, Ye Z. Differential requirement of MKK4 and MKK7 in JNK activation by distinct scaffold proteins. FEBS Lett. 2007 Jan 23;581(2):196-202.
13. Fuchs SY, Adler V, Pincus MR, Ronai Z. MEKK1/JNK signaling stabilizes and activates p53. Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10541-6.
14. Dérijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, Davis RJ. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science. 1995 Feb 3;267(5198):682-5. 
15. Huang CL, Cha SK, Wang HR, Xie J, Cobb MH. WNKs: protein kinases with a unique kinase domain. Exp Mol Med. 2007 Oct 31;39(5):565-73. 
16. Chen W, Yazicioglu M, Cobb MH. Characterization of OSR1, a member of the mammalian Ste20p/germinal center kinase subfamily. J Biol Chem. 2004 Mar 19;279(12):11129-36. 
17. Mukaida N, Wang YY, Li YY. Roles of Pim-3, a novel survival kinase, in tumorigenesis. Cancer Sci. 2011 Aug;102(8):1437-42.
18. Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y, Zhang Z, Lin X, He X. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell. 2002 Mar 22;108(6):837-47. PMID: 11955436.
19. Durzynska I, Xu X, Adelmant G, Ficarro SB, Marto JA, Sliz P, Uljon S, Blacklow SC. STK40 Is a Pseudokinase that Binds the E3 Ubiquitin Ligase COP1. Structure. 2017 Feb 7;25(2):287-294.
20. Kiss-Toth E. Tribbles: 'puzzling' regulators of cell signalling. Biochem Soc Trans. 2011 Apr;39(2):684-7. 

CRISPR Knockout Screening to Identify Protein Kinases That Affect Vasopressin-Stimulated Aqp2 Gene Transcription


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