Abstract
Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY229980
Amino acid identity of the presumed catalytic domains of human PP2C␣ and the corresponding region of Physcomitrella CaMbinding protein phosphatase (PCaMPP) was only 22%, all of the metal-associating residues corresponding to Glu-37, Asp38, Asp-60, Gly-61, Asp-239, and Asp-282 of human PP2C␣ were conserved in PCaMPP (Fig. 1, open arrowheads)
Screening of the moss P. patens cDNA library with radiolabeled CaM resulted in the isolation of a clone encoding a novel Ser/Thr protein phosphatase, PCaMPP
Summary
CaM, calmodulin; ABI, abscisic acidinsensitive; CKII, casein kinase II; GST, glutathione S-transferase; MBP, myelin basic protein; MsCaM, M. sativa calmodulin isoform; PCaMPP, Physcomitrella CaM-binding protein phosphatase; PCM1 and PCM6, potato calmodulin isoforms 1 and 6; PKA, cyclic AMP-dependent protein kinase; PP1, PP2A, PP2B, and PP2C, types 1, 2A, 2B, and 2C phosphoprotein phosphatases; PVDF, polyvinylidene difluoride; DTT, dithiothreitol. Recombinant proteins of PCaMPP were produced, and interaction with CaM and regulation of the phosphatase activity were examined
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