RESUMO
Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.
Assuntos
Oryza/enzimologia , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Biocatálise , Cromatografia de Afinidade , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Oryza/genética , Fosforilação , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/isolamento & purificação , Espectrometria de Massas em Tandem , Técnicas do Sistema de Duplo-HíbridoRESUMO
The rice (Oryza sativa) genome contains 1,429 protein kinases, the vast majority of which have unknown functions. We created a phylogenomic database (http://rkd.ucdavis.edu) to facilitate functional analysis of this large gene family. Sequence and genomic data, including gene expression data and protein-protein interaction maps, can be displayed for each selected kinase in the context of a phylogenetic tree allowing for comparative analysis both within and between large kinase subfamilies. Interaction maps are easily accessed through links and displayed using Cytoscape, an open source software platform. Chromosomal distribution of all rice kinases can also be explored via an interactive interface.
Assuntos
Bases de Dados Factuais , Genoma de Planta , Oryza/enzimologia , Oryza/genética , Fosfotransferases/genética , Filogenia , Motivos de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos de Plantas , Elementos de DNA Transponíveis/genética , DNA de Plantas , Dados de Sequência Molecular , Mutagênese Insercional , Oryza/classificação , Fosfotransferases/metabolismo , Ligação ProteicaRESUMO
Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.
