Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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First published online July 9, 2004; 10.1104/pp.103.035238

Plant Physiology 135:1430-1446 (2004)
© 2004 American Society of Plant Biologists

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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Autophosphorylation and Subcellular Localization Dynamics of a Salt- and Water Deficit-Induced Calcium-Dependent Protein Kinase from Ice Plant1

E. Wassim Chehab, O. Rahul Patharkar, Adrian D. Hegeman, Tahar Taybi and John C. Cushman*

Department of Biochemistry/MS200, University of Nevada, Reno, Nevada 89557–0014 (E.W.C., J.C.C); Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114 (O.R.P); Department of Biochemistry, University of Wisconsin Biotechnology Center, Madison, Wisconsin 53706 (A.H.); and School of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, United Kingdom (T.T.)

A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1–70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K0.5 = 0.15 µM). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 6x His-tagged McCPK1.

1 This work was supported in part by the U.S. Department of Agriculture, National Research Initiative, Competitive Grants Program (grant no. 98–35100–6035 to J.C.C), by the National Science Foundation (grant no. MCB–0114769), and by the Nevada Agricultural Experiment Station (publication no. 03042812 of the Nevada Agricultural Experiment Station).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.035238.

* Corresponding author; e-mail jcushman{at}unr.edu; fax 1–775–784–1650.

Received October 23, 2003; returned for revision April 19, 2004; accepted April 26, 2004.




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