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A novel coiled-coil protein co-localizes and interacts with a calcium-dependent protein kinase in the common ice plant during low-humidity stress

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Abstract

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is induced transiently by salinity and water deficit stress and also McCPK1 undergoes dynamic subcellular localization changes in response to these same stresses. Here we have confirmed that low humidity is capable of causing a drastic change in McCPK1’s subcellular localization. We attempted to elucidate this phenomenon by isolating components likely to be involved in this process. McCAP1 (M. crystallinum CDPK adapter protein 1) was cloned in a yeast two-hybrid screen with a constitutively active McCPK1 as bait. We show that McCPK1 and McCAP1 can interact in the yeast two-hybrid system, in vitro, and in vivo as demonstrated by coimmunoprecipitation experiments from plant extracts. However, McCAP1 does not appear to be a substrate for McCPK1. DsRed–McCAP1 and EGFP–McCPK1 fusions colocalize in epidermal cells of ice plants exposed to low humidity. McCAP1 is homologous to a family of proteins in Arabidopsis with no known function. Computational threading analysis suggests that McCAP1 is likely to be an intermediate filament protein of the cytoskeleton.

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Abbreviations

CDPK:

Calcium-dependent protein kinase

YTH:

Yeast two-hybrid

6× His-tag :

Six histidine tag

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Acknowledgements

The authors would like to thank Dr. Tahar Taybi for providing the McCPK1–SGFP and the McCPK1(Δ1–70)–SGFP constructs. We appreciate all of Asuka Itaya’s help with the microscopy. We are grateful to Dr. Phillip James for providing us with the yeast host stain, PJ69-4A, that was used for this work. We also thank Jen Sheen for providing us with the 35S-SGFP-TYG-nos and the HBT95 constructs, James Carrington for providing the pRTL2 construct, and Felix Randow for a mouse cDNA library. The authors would also like to thank Joan Rowe of University of Nevada Genomics Center for automated DNA sequencing services. This work was supported in part by the U.S. Department of Agriculture, National Research Initiative, Competitive Grants Program Plant Responses to the Environment Program (grant no. 98-35100-6035), by the National Science Foundation 2010 Program (grant no. MCB-0114769), and the Nevada Agricultural Experiment Station, and is published as publication No. 03066928 of the University of Nevada Agricultural Experiment Station.

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  1. Department of Biochemistry/MS200, University of Nevada, 311 Fleischmann Agriculture, Reno, NV, 89557-0014, USA

    John C. Cushman

  2. Department of Molecular Biology, Massachusetts General Hospital, Wellman 11, Boston, MA, 02114, USA

    O. Rahul Patharkar

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  1. O. Rahul Patharkar
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  2. John C. Cushman
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Correspondence to John C. Cushman.

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Patharkar, O.R., Cushman, J.C. A novel coiled-coil protein co-localizes and interacts with a calcium-dependent protein kinase in the common ice plant during low-humidity stress. Planta 225, 57–73 (2006). https://doi.org/10.1007/s00425-006-0330-0

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