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Identification of proteins that interact with catalytically active calcium-dependent protein kinases from Arabidopsis

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Abstract

Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways in plants. Functional characterization of CDPKs is of great interest because they play important roles during growth, development, and in response to a wide range of environmental stimuli. The Arabidopsis genome encodes 34 CDPKs, but very few substrates of these enzymes have been identified. In this study, we exploited the unique characteristics of CDPKs to develop an efficient approach for the discovery of CDPK-interacting proteins. High-throughput, semi-automated yeast two-hybrid interaction screens with two different cDNA libraries each containing 18 million prey clones were performed using catalytically impaired and constitutively active AtCPK4 and AtCPK11 variants as baits. The use of the constitutively active versions of the CPK baits improved the recovery of positive interacting proteins relative to the wild type kinase. Titration of interaction strength by growth under increasing concentrations of 3-aminotriazole (3-AT), a histidine analog and competitive inhibitor of the His3 gene product, confirmed these results. Possible mechanisms for this observed improvement are discussed. The reproducibility of this approach was assessed by the overlap of several interacting proteins of AtCPK4 and AtCPK11 and the recovery of several putative substrates and indicated that yeast two-hybrid screens using constitutively active and/or catalytically impaired forms of CDPK provides a useful tool to identify potential substrates of the CDPK family and potentially the entire protein kinase superfamily.

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Acknowledgments

Special thanks to Mary Ann Cushman and Jennifer Welser for excellent technical assistance. We also thank Alice Harmon, Jeff Harper, and Mike Sussman for their valuable suggestions and fruitful discussions. This work was supported, in part, by grants from the National Science Foundation 2010 Program (MCB-00114769) and the Nevada Agricultural Experiment Station, publication #03087113. This publication was also made possible by NIH Grant Number P20 RR-016464 from the INBRE Program of the National Center for Research Resources through its support of the Nevada Genomics, Proteomics and Bioinformatics Centers.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, 89557-0200, USA

    Yuichi Uno, Miguel A. Rodriguez Milla & John C. Cushman

  2. Department of Plant Resource Science, Graduate School of Agricultural Science, Kobe University, Rokko, Kobe, 657-8501, Japan

    Yuichi Uno

  3. Departamento de Biotecnologia/Edificio Z, INIA, Ctra. de la Coruña Km. 7,5, 28040, Madrid, Spain

    Miguel A. Rodriguez Milla

  4. Molecular Interaction Facility/Biotechnology Center, University of Wisconsin, Madison, WI, 53706, USA

    Eileen Maher

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  1. Yuichi Uno
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  2. Miguel A. Rodriguez Milla
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  4. John C. Cushman
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Correspondence to John C. Cushman.

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Communicated by I. Stagljar.

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Uno, Y., Rodriguez Milla, M.A., Maher, E. et al. Identification of proteins that interact with catalytically active calcium-dependent protein kinases from Arabidopsis . Mol Genet Genomics 281, 375–390 (2009). https://doi.org/10.1007/s00438-008-0419-1

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