Abstract
BackgroundModulation 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.ResultsHere 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0900-7) contains supplementary material, which is available to authorized users.
Highlights
Modulation of protein activity by phosphorylation through kinases and subsequent dephosphorylation by phosphatases is one of the most prominent cellular control mechanisms
Generation of an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A (CDKA;1) Arabidopsis CDKA;1 shares a high degree of sequence similarity with human Cyclin-dependent kinase 1 (Cdk1), Cyclin-dependent kinase 2 (Cdk2), and Cdk3 as well as the yeast Cdc2 and CDC28 kinases (Additional file 1: Figure S1A)
When we modeled CDKA;1 onto a known crystal structure of human Cdk2, all of the important structural elements of Cdk2 could be matched in CDKA;1, e.g. the T-loop and the P-loop, in accordance with previous reports showing that the molecular mechanistics of CDKA;1 function are conserved (Additional file 1: Figure S1B) [31,32,33]
Summary
Modulation of protein activity by phosphorylation through kinases and subsequent dephosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Substantial work in yeast and animal model systems has shown that high kinase activity levels are in particular required to promote the transition from a gap phase (G1) into S phase where the nuclear DNA becomes replicated and from a second gap phase (G2) into M phase (mitosis) during which the chromosomes are distributed to the newly forming daughter cells. At these two major control points, CDK-cyclin complexes phosphorylate a plethora of target proteins. Currently very little is known about the molecular basis of the integration of the cell cycle with other cell-physiological processes
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