According to the similarity of the amino acid sequences in their catalytic domains, eukaryotic protein kinases have been classified into the five main groups: 'AGC', 'CaMK', 'CMGC', 'PTK' and 'other'. The AGC group, represented by the cyclic nucleotide-dependent kinases (PKA and PKG), the calcium-phospholipid-dependent kinases (PKC) and the ribosomal S6 protein kinases, are poorly characterized in plants except for a few cases. In this study, in order to gain a better understanding of plant protein kinases in the AGC group, three cDNAs encoding novel protein kinases, RsNdr1 and RsNdr2a/b, were cloned from radish and characterized by molecular and biochemical methods.The deduced amino acid sequences of RsNdr1 and RsNdr2a/b contained all 12 conserved catalytic subdomains which are characteristic of the eukaryotic Ser/Thr protein kinases. A cell lysate from E. coli overexpressing RsNdr1 fusion protein had protein kinase activity toward a conventional protein substrate (myelin basic protein), whereas that from E. coli harboring a fusion plasmid encoding kinasedead RsNdr1 or RsNdr2 did not show any protein kinase activity. A phylogenetic tree for 17 protein kinases from various organisms showed that the RsNdrs are more closely related to the protein kinases in a particular subgroup of the 'AGC' (fungal cot1 -like and animal Ndr kinases) than to the authentic 'AGC' protein kinases, such as PKA, PKC or ribosomal S6 kinase.
In order to gain a better understanding of the nature of nuclear dbf-related (Ndr) protein kinase homologs in monocots, three cDNA clones were isolated from common wheat (Triticum aestivum) by data mining of expressed sequence tag (EST) databases, and their primary structure was determined by reverse transcriptionpolymerase chain reaction (RT-PCR), 5′-rapid amplification of cDNA ends (RACE), and nucleotide sequencing. Deduced amino acid sequences of the cDNA clones contained all 12 highly conserved subdomains of the eukaryotic Ser/Thr protein kinase, including the ATP-binding site (in subdomain I-II) and the Ser/Thr protein kinase active-site (in subdomain VI). The sequences also contained an insert of 56 amino acids between subdomains VII and VIII, and three conserved Ser/Thr residues, being characteristic of the Ndr family of eukaryotic protein kinases. A sequence comparison among cDNAs from wheat and those from ancestral diploid species (T. boeoticum, Aegilops speltoides and Ae. squarrosa) revealed that at least three homeologous genes were expressed in hexaploid wheat. The results confirmed the usefulness of current EST databases including KOMUGI for gene cloning of wheat, since the use of specific primers designed with information about EST sequences has considerably facilitated the cloning of rare cDNAs such as Ndr.
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