Regulation of the dual specificity protein phosphatase, DsPTP1, through interactions with calmodulin.
Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.
Yoo
Jae Hyuk
JH
Division of Applied Life Science, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea.
Cheong
Mi Sun
MS
Park
Chan Young
CY
Moon
Byeong Cheol
BC
Kim
Min Chul
MC
Kang
Yun Hwan
YH
Park
Hyeong Cheol
HC
Choi
Man Soo
MS
Lee
Ju Huck
JH
Jung
Won Yong
WY
Yoon
Hae Won
HW
Chung
Woo Sik
WS
Lim
Chae Oh
CO
Lee
Sang Yeol
SY
Cho
Moo Je
MJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000737:chemistry; D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D054637:Dual-Specificity Phosphatases; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009943:Organophosphorus Compounds / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction
D000814:Aniline Compounds; D029681:Arabidopsis Proteins; D002147:Calmodulin; D018076:DNA, Complementary; D009943:Organophosphorus Compounds; D010455:Peptides; D019000:Phosphotyrosine; C068798:4-aminophenylphosphate; D005982:Glutathione Transferase; D054645:Protein Phosphatase 1; C481271:DsPTP1 protein, Arabidopsis; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases; D010727:Phosphoric Diester Hydrolases; D002118:Calcium