Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1.
BACKGROUND
Modulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many - if not all - molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase.


RESULTS
Here we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates.


CONCLUSIONS
The here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology.
Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS - UPR2357, Université de Strasbourg, F-67084, Strasbourg, France.|Trinationales Institut für Pflanzenforschung, F-67084, Strasbourg Cedex, France.|Present address: RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan.
Hirofumi
H
Harashima
Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS - UPR2357, Université de Strasbourg, F-67084, Strasbourg, France.|Trinationales Institut für Pflanzenforschung, F-67084, Strasbourg Cedex, France.|Present address: Leibniz Institute of Plant Biochemistry (IPB), Independent Junior Research Group on Protein Recognition and Degradation, Weinberg 3, D-06120, Halle, (Saale), Germany.
Nico
N
Dissmeyer
Plateforme protéomique Strasbourg Esplanade, Institut de Biologie Moléculaire et Cellulaire FRC1589-CNRS, F-67084, Strasbourg, France.
Philippe
P
Hammann
Plant Proteomics Research Unit, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
Yuko
Y
Nomura
Max Planck Institute for Plant Breeding Research, Basic Immune System of Plants / Protein Mass Spectrometry, Carl-von-Linne-Weg 10, 50829, Cologne, Germany.
Katharina
K
Kramer
Plant Proteomics Research Unit, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.|Max Planck Institute for Plant Breeding Research, Basic Immune System of Plants / Protein Mass Spectrometry, Carl-von-Linne-Weg 10, 50829, Cologne, Germany.
Hirofumi
H
Nakagami
Department of Molecular Mechanisms of Phenotypic Plasticity, Institut de Biologie Moléculaire des Plantes du CNRS, IBMP-CNRS - UPR2357, Université de Strasbourg, F-67084, Strasbourg, France. arp.schnittger@uni-hamburg.de.|Trinationales Institut für Pflanzenforschung, F-67084, Strasbourg Cedex, France. arp.schnittger@uni-hamburg.de.|Department of Developmental Biology, University of Hamburg, Biozentrum Klein Flottbek, Ohnhorststr. 18, D-22609, Hamburg, Germany. arp.schnittger@uni-hamburg.de.
Arp
http://orcid.org/0000-0001-7067-0091
A
Schnittger
Arabidopsis; Cell cycle; Kinase; Mitosis; Phosphorylation; Substrate
Plant Physiol. 1973 Jun;51(6):1127-32
16658478
Bioorg Med Chem Lett. 2008 Dec 15;18(24):6369-73
18996007
Proc Natl Acad Sci U S A. 1982 Apr;79(7):2231-5
6285374
Gene. 1991 Sep 15;105(2):159-65
1937013
Adv Enzyme Regul. 2010;50(1):375-99
20045433
Annu Rev Cell Dev Biol. 1997;13:261-91
9442875
J Biol Chem. 1979 Nov 10;254(21):11148-53
500632
FEBS Lett. 2006 Feb 6;580(3):904-11
16438971
J Biol Chem. 2003 Apr 25;278(17 ):14926-35
12594221
Plant Mol Biol. 2003 Nov;53(4):423-42
15010610
J Med Chem. 2002 Aug 1;45(16):3381-93
12139449
Annu Rev Cell Dev Biol. 2001;17:405-33
11687495
Methods Mol Biol. 2011;779:7-52
21837559
Cell Death Differ. 2009 May;16(5):738-48
19229245
Methods Mol Biol. 2011;779:245-57
21837571
Plant Physiol. 2006 Oct;142(2):564-73
16891550
Curr Protein Pept Sci. 2000 Sep;1(2):209-35
12369917
Biochem Biophys Res Commun. 1974 Jun 18;58(4):960-7
4275862
Cell Cycle. 2012 Apr 15;11(8):1626-33
22487684
Philos Trans R Soc Lond B Biol Sci. 2012 Sep 19;367(1602):2619-39
22889912
Trends Mol Med. 2012 Jun;18(6):337-47
22578879
Curr Opin Plant Biol. 2010 Feb;13(1):66-74
19963429
Bioinformatics. 2002 Sep;18(9):1274-5
12217924
FEBS Lett. 2006 Jan 9;580(1):336-40
16376885
PLoS Genet. 2012;8(7):e1002865
22844260
Annu Rev Cell Dev Biol. 2012;28:59-87
22578140
FEBS Lett. 1997 Aug 4;412(3):446-52
9276444
Nature. 2005 Mar 3;434(7029):104-8
15744308
Nucleic Acids Res. 2010 Jan;38(Database issue):D828-34
19880383
Proc Natl Acad Sci U S A. 2012 Apr 24;109(17):E1019-27
22474407
Plant Methods. 2012 Jun 28;8(1):23
22741569
Chem Soc Rev. 2011 Mar;40(3):1211-23
21152652
Plant J. 2007 Nov;52(3):435-48
17764501
Nat Cell Biol. 1999 Nov;1(7):438-43
10559988
Nature. 1995 Jul 27;376(6538):313-20
7630397
Nucleic Acids Res. 2008 Jan;36(Database issue):D1015-21
17984086
Plant Mol Biol. 2007 Dec;65(6):817-27
17924062
Plant J. 2009 Nov;60(4):679-90
19674406
J Biol Chem. 2009 May 29;284(22):15289-98
19332544
Plant Physiol. 2008 Feb;146(2):351-67
18156295
Science. 2009 Sep 25;325(5948):1682-6
19779198
Environ Microbiol. 2011 Sep;13(9):2518-35
21854516
Mol Cell. 2009 Jan 16;33(1):53-63
19150427
PLoS Genet. 2011 Mar;7(3):e1001348
21455289
Plant Cell. 1993 Dec;5(12):1711-23
8305869
Plant Cell. 1991 May;3(5):531-40
1840925
Nat Biotechnol. 2008 Dec;26(12):1367-72
19029910
Plant Cell. 2012 Oct;24(10 ):4083-95
23104828
Plant Cell. 2009 Nov;21(11):3641-54
19948791
Cell Cycle. 2011 May 1;10(9):1378-84
21552008
ACS Chem Biol. 2011 May 20;6(5):492-501
21291269
Plant Cell. 2007 Mar;19(3):972-85
17369369
Protein Sci. 1993 Jan;2(1):31-40
8382990
Nature. 2010 Dec 23;468(7327):1074-9
21179163
Nature. 2000 Sep 21;407(6802):395-401
11014197
Nature. 2003 Oct 23;425(6960):859-64
14574415
Plant J. 2012 Apr;70(2):348-56
22098373
ACS Chem Biol. 2007 May 22;2(5):299-314
17518431
Nat Methods. 2007 Jun;4(6):511-6
17486086
Chembiochem. 2009 Jun 15;10(9):1519-26
19437469
Nature. 2000 Dec 14;408(6814):796-815
11130711
Anal Biochem. 2005 Aug 15;343(2):313-21
15993367
Mol Cell Biochem. 1980 Apr 18;30(2):111-27
6991912
Curr Biol. 2013 Oct 21;23(20):1969-78
24120637
Nat Methods. 2005 Jun;2(6):435-41
15908922
Cell Cycle. 2012 Apr 15;11(8):1621-5
22456336
Nat Genet. 2006 Jan;38(1):63-7
16311592
Bioorg Med Chem Lett. 2009 Feb 1;19(3):908-11
19097791
FEBS Lett. 1999 Mar 5;446(1):182-8
10100639
Dev Cell. 2012 May 15;22(5):1030-40
22595674
Top Curr Chem. 2013;329:179-201
22752581
Curr Opin Plant Biol. 2010 Jun;13(3):280-87
20097120
Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
12824332
Plant J. 2006 Aug;47(4):532-46
16813576
Plant Physiol. 2011 Oct;157(2):659-72
21803861
Methods Mol Biol. 2011;779:259-71
21837572
Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1442-7
18234856
J Biol Chem. 2011 Aug 19;286(33):28794-801
21697089
Methods Mol Biol. 2011;779:93-138
21837563
BMC Plant Biol. 2016 Feb 24;16:48
26912131
Plant Physiol. 2002 Mar;128(3):812-21
11891238
J Mol Biol. 1993 Dec 5;234(3):779-815
8254673
Methods Mol Biol. 2011;779:65-78
21837561