A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57.
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 6xHis-Ki-1/57(122-413) and 6xHis-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 beta subunit as template. Our model suggests the family-typical seven-bladed beta-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.
Nery
Flávia C
FC
Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Rua Giuseppe Máximo Scolfaro 10.000, C.P. 6192, 13084-971 Campinas SP, Brazil.
Bressan
Gustavo C
GC
Alborghetti
Marcos R
MR
Passos
Dario O
DO
Kuniyoshi
Taís M
TM
Ramos
Carlos H I
CH
Oyama
Sergio
S
Kobarg
Jörg
J
D002942:Circular Dichroism; D003198:Computer Simulation; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D017730:Ki-1 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism; D064546:Protein Kinase C beta; D017434:Protein Structure, Tertiary; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods*; D020798:Two-Hybrid System Techniques
D044387:GTP-Binding Protein beta Subunits; D017730:Ki-1 Antigen; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D011493:Protein Kinase C; D064546:Protein Kinase C beta