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SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION

Large-Scale Arabidopsis Phosphoproteome Profiling Reveals Novel Chloroplast Kinase Substrates and Phosphorylation Networks^1,[W]

Department of Biology, Eidgenössische Technische Hochschule Zurich, 8092 Zurich, Switzerland (S.R., G.M., K.B., B.G., A.E., J.G., W.G., S.B.); Functional Genomics Center Zurich, 8057 Zurich, Switzerland (B.G., J.G.); and Institute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland (A.E.)

We have characterized the phosphoproteome of Arabidopsis (Arabidopsis thaliana) seedlings using high-accuracy mass spectrometry and report the identification of 1,429 phosphoproteins and 3,029 unique phosphopeptides. Among these, 174 proteins were chloroplast phosphoproteins. Motif-X (motif extractor) analysis of the phosphorylation sites in chloroplast proteins identified four significantly enriched kinase motifs, which include casein kinase II (CKII) and proline-directed kinase motifs, as well as two new motifs at the carboxyl terminus of ribosomal proteins. Using the phosphorylation motifs as a footprint for the activity of a specific kinase class, we connected the phosphoproteins with their putative kinases and constructed a chloroplast CKII phosphorylation network. The network topology suggests that CKII is a central regulator of different chloroplast functions. To provide insights into the dynamic regulation of protein phosphorylation, we analyzed the phosphoproteome at the end of day and end of night. The results revealed only minor changes in chloroplast kinase activities and phosphorylation site utilization. A notable exception was ATP synthase β-subunit, which is found phosphorylated at CKII phosphorylation sites preferentially in the dark. We propose that ATP synthase is regulated in cooperation with 14-3-3 proteins by CKII-mediated phosphorylation of ATP synthase β-subunit in the dark.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org)are: Wilhelm Gruissem (wgruissem{at}ethz.ch) and Sacha Baginsky (sbaginsky{at}ethz.ch).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.138677

^* Corresponding author; e-mail sbaginsky{at}ethz.ch.

Received March 13, 2009; accepted April 14, 2009; published April 17, 2009.

This article has been cited by other articles:

				S. Baginsky and W. Gruissem The Chloroplast Kinase Network: New Insights from Large-Scale Phosphoproteome Profiling Mol Plant, November 1, 2009; 2(6): 1141 - 1153. [Abstract] [Full Text] [PDF]

				P. Durek, R. Schmidt, J. L. Heazlewood, A. Jones, D. MacLean, A. Nagel, B. Kersten, and W. X. Schulze PhosPhAt: the Arabidopsis thaliana phosphorylation site database. An update Nucleic Acids Res., October 30, 2009; (2009) gkp810v1. [Abstract] [Full Text] [PDF]