Expression and characterization of PKL01, an Ndr kinase homolog in Lotus japonicus.
We isolated cDNA clones for novel protein kinases by expression cloning from Lotus japonicus. The LNZ001, one of the isolated clones, encodes a protein of 547 amino acids with a predicted molecular weight of 63,349. Since the protein contains 12 highly conserved subdomains specific to Ser/Thr protein kinases, we designated it PKL01. When homology searches based on the PKL01 sequence were carried out, the protein was found to show sequence homology with nuclear Dbf2-related kinases (Ndr kinases). When PKL01 was produced using an Escherichia coli expression system and purified to homogeneity, it underwent intermolecular autophosphorylation. The major autophosphorylation site was identified as Ser-317 by using various point mutants, and phosphorylation at this site was found to be critical for the kinase activity. PKL01 was found to be widely distributed in the leaves, stems, roots and root nodules by northern hybridization experiments. When endogenous substrates were screened using fractionated preparations from various parts of plants, PKL01 preferentially phosphorylated basic proteins in tissue extracts. These results suggest that PKL01 is an Ndr kinase homolog in L. japonicus and may be involved in the regulation of cellular functions through phosphorylation of basic protein substrates such as histones.
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan. kamesita@ag.kagawa-u.ac.jp
Isamu
I
Kameshita
Sachiko
S
Shimomura
Kazushi
K
Nishio
Noriyuki
N
Sueyoshi
Tetsuyuki
T
Nishida
Mika
M
Nomura
Shigeyuki
S
Tajima
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006657:Histones / Q000378:metabolism; D006801:Humans; D000070116:Lotus / Q000235:genetics*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D006657:Histones; D010940:Plant Proteins; D017346:Protein Serine-Threonine Kinases; C491614:STK38 protein, human