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.

Involvement of Ca(2+)/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus.

Although multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) are widely distributed in animal cells, the occurrence of CaM-kinases in the basidiomycetous mushroom has not previously been documented. When the extracts from various developmental stages from mycelia to the mature fruiting body of Coprinus cinereus were analyzed by Western blotting using Multi-PK antibodies, which had been generated to detect a wide variety of protein serine/threonine kinases (Ser/Thr kinases), a variety of stage-specific Ser/Thr kinases was detected. Calmodulin (CaM) overlay assay using digoxigenin-labeled CaM detected protein bands of 65 kDa, 58 kDa, 46 kDa, 42 kDa, and 38 kDa only in the presence of CaCl(2), suggesting that these bands were CaM-binding proteins. When the CaM-binding fraction was prepared from mycelial extract of C. cinereus by CaM-Sepharose and analyzed with Multi-PK antibodies, two major immunoreactive bands corresponding to 65 kDa and 46 kDa were detected. CaM-binding fraction, thus obtained, exhibited Ca(2+)/CaM-dependent protein kinase activity toward protein substrates such as histones. These CaM-kinases were found to be highly expressed in the actively growing mycelia, but not in the resting mycelial cells. Mycelial growth was enhanced by the addition of CaCl(2) in the culture media, but inhibited by the addition of EGTA or trifluoperazine, a potent CaM inhibitor. This suggested that CaM-dependent enzymes including CaM-kinases play crucial roles in mycelial growth of basidiomycete C. cinereus.

Expression cloning of a variety of novel protein kinases in Lotus japonicus.

To investigate protein kinases expressed in Lotus japonicus, a cDNA expression library of the root-nodule of L. japonicus was immunologically screened with monoclonal antibodies directed to a highly conserved region in protein serine/threonine kinases (Ser/Thr kinases). Among 178 positive clones obtained from the lambdaZAPII cDNA library, 164 clones were found to encode novel proteins possessing the subdomain VIB sequences characteristic of Ser/Thr kinases. By phylogenetic analysis on the basis of deduced amino acid sequences, the isolated clones could be classified into five different families of Ser/Thr kinases : the SnRK family, GSK-3 family, Ndr kinase family, Ark family, and receptor kinase family. These results suggest that this expression cloning using the kinase-specific antibodies will provide new clues for investigations of a wide variety of known and novel protein kinases in higher plants.