Involvement of the residues of GSKIP, AxinGID, and FRATtide in their binding with GSK3beta to unravel a novel C-terminal scaffold-binding region.
The specificity and regulation of GSK3beta are thought to involve in the docking interactions at core kinase domain because of the particular amino acid residues. Recent X-ray diffraction studies illuminated the relative binding residues on AxinGID and FRATtide for GSK3beta docking and appeared that GSK3beta Val267Gly (V267G) and Tyr288Phe (Y288F) could distinguish the direct interaction between AxinGID and FRATtide. In order to explore the mode that involved the binding of GSKIP to GSK3beta and compare it with that of AxinGID and FRATtide, we pinpointed the binding sites of GSKIP to GSK3beta through the single-point mutation of four corresponding sites within GSK3beta (residues 260-300) as scaffold-binding region I (designated SBR-I(260-300)). Our data showed that these three binding proteins shared similar binding sites on GSK3beta. We also found that the binding of GSK3beta V267G mutant to GSKIP and AxinGID, but not that of Y288F mutant (effect on FRATtide), was affected. Further, based on the simulation data, the electron-density map of GSKIPtide bore closer similarity to the map AxinGID than to that of FRATtide. Interestingly, many C-terminal helix region point-mutants of GSK3beta L359P, F362A, E366K, and L367P were able to eliminate the binding with FRATtide, but not AxinGID or GSKIP. In addition, CABYR exhibited a unique mode in binding to C-terminal helix region of GSK3beta. Taken together, our data revealed that in addition to the core kinase domain, SBR-I(260-300), another novel C-terminus helix region, designated SBR-II(339-383), also appeared to participate in the recognition and specificity of GSK3beta in binding to other specific proteins.
Howng
Shen-Long
SL
Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC.
Hwang
Chi-Ching
CC
Hsu
Chia-Yi
CY
Hsu
Meng-Yu
MY
Teng
Chun-Yen
CY
Chou
Chia-Hua
CH
Lee
Mei-Feng
MF
Wu
Chia-Hung
CH
Chiou
Shean-Jaw
SJ
Lieu
Ann-Shung
AS
Loh
Joon-Khim
JK
Yang
Chia-Ning
CN
Lin
Chan-Shing
CS
Hong
Yi-Ren
YR
D000595:Amino Acid Sequence; D060466:Axin Protein; D015153:Blotting, Western; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D051176:beta Catenin / Q000378:metabolism
D060466:Axin Protein; C514503:GSKIP protein, human; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012097:Repressor Proteins; D051176:beta Catenin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3