Molecular analysis of the interaction between cardosin A and phospholipase D(alpha). Identification of RGD/KGE sequences as binding motifs for C2 domains |
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Authors: | Simões Isaura Mueller Eva-Christina Otto Albrecht Bur Daniel Cheung Alice Y Faro Carlos Pires Euclides |
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Affiliation: | Departamento de Biologia Molecular e Biotecnologia, Centro de Neurociências e Biologia Celular, Universidade de Coimbra and Departamento de Bioquímica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Portugal. |
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Abstract: | Here we report the identification of phospholipase Dalpha as a cardosin A-binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull-down assays. To investigate the structural and molecular determinants involved in the interaction, pull-down assays with cardosin A and various glutathione S-transferase-fused phospholipase Dalpha constructs were performed. Results revealed that the C2 domain of phospholipase Dalpha contains the cardosin A-binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge-wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase Dalpha can act as a cardosin A-binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE-recognition domains. |
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