Identification of Putative Plant PTKs by Comparative Proteome Bioinformatics

Most of the >900 putative protein kinases detected in silico in the proteome of A. thaliana have been tentatively assigned to the Ser/Thr class [37]. However, putative specificity reported in annotated sequences is commonly inferred by similarity from other sequences; and this is inappropriate in the case of eukaryotic protein kinases, which are known to share a highly conserved catalytic domain while residue and substrate phosphorylation specificities are determined by motif variation and by noncatalytic regions. Given that experimental evidence on phosphorylation specificity concerns only a limited subset of protein kinases from a given proteome, and considering that in animals PTKs represent a minor fraction of the kinase complement, the presence of PTKs and/or PTK-like kinases among as yet uncharacterized products of plant kinase genes cannot be excluded a priori.

Although no classical PTK has hitherto been cloned from plants, a comparative bioinformatic analysis identified a complement of putative PTKs in A. thaliana [38]. Such putative kinases were identified by screening the whole proteome with highly specific sequence markers. In fact, the protein kinase domain is too conserved among Ser/Thr and Tyr kinases to allow specific detection of putative PTKs by homology search. This problem was overcome using as sequence probes patterns PS00108 and PS00109 from the well-known database PROSITE, which contains "similarity-independent" amino acid signatures. In order to further improve the extraction of putative PTKs, Carpi et al. [38] took into account the problem of true hits, endowed with the proband activity but showing a degenerate signature (FN hits) and screened the Arabidopsis proteome using also slightly degenerate PROSITE signatures as sequence probes. Positive hits showing either canonical or degenerate patterns were then analyzed to discard FP hits corresponding to nonkinase proteins or nonfunctional kinases (e.g., lacking one or more subdomains, or mutated at residues crucial to the enzyme activity). Then, all putative PTK or DSK sequences were further analyzed to group them based on conserved or specific moieties, and the expression of corresponding genes in green tissues was assessed. Strategy that led to the identification of a complement of putative PTKs and DSKs in Arabidopsis is summarized in Figure 34.1.

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