A stress-induced calcium-dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two-component pseudo-response regulator.
McCDPK1 is a salinity- and drought-induced calcium-dependent protein kinase (CDPK) isolated from the common ice plant, Mesembryanthemum crystallinum. A yeast two-hybrid experiment was performed, using full-length McCDPK1 and truncated forms of McCDPK1 as baits, to identify interacting proteins. A catalytically impaired bait isolated a cDNA clone encoding a novel protein, CDPK substrate protein 1 (CSP1). CSP1 interacted with McCDPK1 in a substrate-like fashion in both yeast two-hybrid assays and wheat germ interaction assays. Furthermore, McCDPK1 was capable of phosphorylating CSP1 in vitro in a calcium-dependent manner. Our results demonstrate that the use of catalytically impaired and unregulated CDPKs with the yeast two-hybrid system can accelerate the discovery of CDPK substrates. The deduced CSP1 amino acid sequence indicated that it is a novel member of a class of pseudo-response regulator-like proteins that have a highly conserved helix-loop-helix DNA binding domain and a C-terminal activation domain. McCDPK1 and CSP1 co-localized to nuclei of NaCl-stressed ice plants. Csp1 transcript accumulation was not regulated by NaCl or dehydration stress. Our results strongly suggest that McCDPK1 may regulate the function of CSP1 by reversible phosphorylation.
Patharkar
O R
OR
Department of Biochemistry/MS200, 311B Fleischmann Agriculture, University of Nevada, Reno, NV 89557-0014, USA.
Cushman
J C
JC
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology*; D002467:Cell Nucleus / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D049452:Green Fluorescent Proteins; D018257:Helix-Loop-Helix Motifs; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D010936:Plant Extracts / Q000378:metabolism; D010940:Plant Proteins; D010944:Plants / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014908:Triticum; D020798:Two-Hybrid System Techniques
C419902:CSP1 protein, Mesembryanthemum crystallinum; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D010936:Plant Extracts; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D012965:Sodium Chloride; D011494:Protein Kinases; D002118:Calcium