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Flávia C. Nery, Gustavo C. Bressan, Marcos R. Alborghetti, Dario O. Passos, Taís M. Kuniyoshi, Carlos H.I. Ramos, Sergio Oyama, Jörg Kobarg

A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57

Keywords: circular dichroism, emission fluorescence, molecular modeling, protein-protein interaction, regulatory proteins, surface tryptophans

Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6×His-Ki-1/57(122–413) and 6×His-Ki-1/57(264–413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein ? subunit as template. Our model suggests the family-typical seven-bladed ?-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 387, 05/2006
Pages: 577 - 582

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