Transcript 投影片 1

GSKIP, shares a homology of Axin GID domain and functions as a negative
regulator of GSK3beta
4P-C-110
Tai-Shan Cheng1, He-Yen Chou1, Shen-Long Howng2, Yun-Ling Hsiao1, Ann-Shung Lieu2, Joon-Khim Loh2, Shiuh-Lin Hwang2, Ching-Chih Lin1,3, ChingMei Hsu3, Chihuei Wang4, Chu-I Lee5, Pei-Jung Lu6, Chen-Kung Chou7, Chi-Ying Huang8 and Yi-Ren Hong1,9
1Graduate Institute of Biochemistry; 2Department of Neurosurgery, Kaohsiung Medical University Hospital; 3 Department of Biological sciences, National Sun YatSen University, Kaohsiung; 4Department of Biotechnology; Kaohsiung Medical University; 5Department of Medical Technology, Fooyin University, Kaohsiung;
6Kaohsiung Veterans General Hospital, Kaohsiung; 7Department of Life Sciences, Chang-Gung University, Taoyuan; 8National Health Research Institutes, Taipei
and 9Department of Clinical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, R.O.C
Abstract
Although prominent FRAT/GBP exhibits limited homology with Axin, the binding sites on
GSK3 for FRAT/GBP and Axin may overlap to prevent the effect of FRAT/GBP to stabilize
b-catenin in Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein,
GSK3b interaction protein (GSKIP), which binds to GSK3beta. We have defined a 25-aminoacid residues region in the C-terminal of GSKIP that is highly similar to the GSK3beta
interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that
GSKIP is a good GSK3beta substrate and both full-length and a C-terminal fragment of
GSKIP can block phosphorylation of primed and non-primed substrates in different fashions.
Similar to Axin GID381-405 and FRATtide, synthesized GSKIPtide is also shown to compete
with and/or block the phosphorylation of Axin and beta-catenin by GSK3beta. Furthermore,
our data indicate that overexpression of GSKIP induces beta-catenin accumulation in the
cytoplasm and nucleus as visualized by immunofluorescence. A functional assay also
demonstrates that GSKIP transfected cells show a significant effect on the transactivity of
Tcf-4. Collectively, we define GSKIP as a naturally occurring protein that shares a homology
with the GID domain of Axin and is able to negatively regulate GSK3beta of the Wnt
signaling pathway.
Results
Figure 5. GSKIP
GSK3b activity.
inhibits
Four reactions were analyzed in the presence of assay
mixtures containing recombinant GST-Axin275-510; GST-bcatenin; His-tagged Tau or glycogen synthase (GS) and
GSK3b. (A) GST-Axin275-510; (B) GST-b-catenin (in the
presence of Axin275-510); (C) His-tagged Tau; (D) GS. All
reactions contained GSKIP at various doses (0, 0.2, 1, 5 µM).
Arrow indicates phosphorylated GST-Axin (275-510 aa), Histagged Tau, and GST-b-catenin. Arrowhead indicates
phosphorylated GSKIP. In (A) mark C (left panel lane 5)
indicates the His-Tag-vector protein, which acts as a negative
control. GID320-429 (right panel) contains various doses of the
peptide (0, 0.2, 1 mM) and acts as a positive control.
Figure 6. Synthesized GSKIPtide acts as an
inhibitor.
Four reactions were analyzed in the presence of assay mixtures
containing recombinant GST-Axin275-510; GST-b-catenin; His-tagged
Tau or glycogen synthase (GS) and GSK3b. (A) GST-Axin275-510; (B)
GST-b-catenin (in the presence of Axin275-510); (C) His-tagged Tau; (D)
GS. All reactions contained various doses of GSKIPtide (0, 20, 100,
500 mM).
Figure 7. GSKIP causes b-catenin accumulation in the
cytoplasm and nucleus and activates the reporter systems.
(A) GSKIP induces b-catenin accumulation in the cytoplasm and
nucleus as visualized by immunofluorescence. HeLa cells were
co-transfected with GSKIP, GSKIP(L130P) or pIRES vector,
together with pEGFP. GSKIP expression is indicated in the
transfected cells by green. b-catenin is stained with rhodamineconjugated secondary antibody and is red. Nuclei are stained
with DAPI (blue). (B) HEK293 cells were transfected with 2µg wt
b-catenin plus pIRES-GSKIP, pIRES-GSKIP (L130P) or pIRES
vector and compare with a positive control, b-catenin (41;45). bcatenin accumulation in the presence of GSKIP in HEK293 cells.
b-catenin was detected by Western blotting (left panel). Fold
induction indicates transcriptional activity compared with pIRES
vector control plasmid (right panel). (C) HEK293 cells were
transfected with increasing concentration of GSKIP as indicated.
Each value represents the mean ± SD of three separate
experiments. Statistically significant differences as determined
by a Student’s t test: * p<0.005, ** p<0.0005 versus control.
Figure 2. A 25-aa residue sequence of
GSKIP is high similarly to the GID of Axin.
Figure
1.
Protein
sequence
alignment of GSKIP and its
subcellular localization.
directly
(A) Serial deletion mutants of GSKIP indicating interaction with
GSK3b. “+” indicates strong interaction, “–” indicates no interaction.
(B) Amino acid sequence of GSKIP115-139, which similar to highly
conserved region of Axin1381-405 and Axin2363-387. Amino acid
similarities between GSKIP and Axin proteins are highlighted in
shadow. Amino acid identities are highlighted in bold. (C)
GSKIP(L130P) mutation prevents GSK3b association with GSKIP.
Growth indicates a positive interaction.
(A) Schematic representation of the GSKIP and its
domains. The genomic organization of GSKIP is shown
with the three exons. (B) Protein sequence alignment of
seven species. Note that C-terminal 109-139 aa of
GSKIP (GSKIP109-139) contains a highly conserved
region across all seven species. (C) Northern blotting
analysis of GSKIP expression in various human tissues.
The membrane contained ~2mg of poly(A+) mRNA from
each tissue. Hybridization was done using [a-32P]labeled cDNA probe for the full-length GSKIP with
human b-actin as a control. (D) Localization of GSKIP.
Summary & Conclusion
We reports the isolation by Y2H screening of a gene termed GSKIP(GSK3b
interaction protein) encoding a protein that interacts with GSK3b and is a
substrate for this kinase.
The C-terminal region of GSKIP possesses a 25-aa region similar to the GID381-405
of Axin. This region is required for GSK3b binding.
Expression of GSKIP inhibits the capacity of GSK-3 to phosphorylate certain
substrates such as b-catenin.
Figure 4. GSK3b phosphorylates GSKIP at
S109 and T113.
Figure 3. GSKIP interacts with GSK3b in
vivo and in vitro.
(A) Coomassie blue staining of GSKIP constructs. (B) GST pull
down analysis of GSKIP with GSK3b. The two fusion proteins
were tested for co-eluted from glutathione-Sepharose 4B, which
would indicate interaction between GSKIP (full-length) or
GSKIP109-139 and GSK3b. (C) Coimmunoprecipitation of GSKIP
with GSK3b. HEK293 cells were cotransfected with pCMV-FlagGSK3b and/or pcDNA-GSKIP or pcDNA-GSKIP(L130P) or
pcDNA vector. Immunoprecipitation (IP) was performed with antiFlag antibody. Western-blotting (WB) was performed using antiHA antibody.“ ★” indicates the GSKIP signal.
(A) The kinase assay was performed using purified GSKIP, GSKIP1-108,
GSKIP109-139, GSKIP(L130P) and GSK3b. (B) Schematic diagram of three
putative phosphorylation sites (Ser109; Thr113; Ser115) and GSK3b
interaction domain (115-139aa) of GSKIP109-139. (C) To perform kinase
assays, we tested wt (lane 1); S109A (lane 2); T113A (lane 3); S115A (lane
4); S109A/ T113A (lane 5); and S109A/ T113A /S115A (lane 6) for
GSKIP109-139 phosphorylation by GSK3b. GSKIP (full-length), wt (lane 7);
S109A (lane 8); T113A (lane 9); S115A (lane 10); S109A/ T113A (lane 11)
and S109A/ T113A /S115A (lane 12) also underwent phosphorylation by
GSK3b. Below boxes show equal amount were added of GSKIP109-139 and
GSKIP (full-length) detected by Western blot as control. Phosphorylation
quantified by BIO-PROFIL Bio-1D. Data are presented as the mean ± S.E.
from three independent experiments, each performed in duplicate.
The function of GSKIP is also similar to FRAT/GBP (despite the lack of sequence
similarity between FRATtide and GSKIPtide), our results indicate that GSKIP and
GSKIPtide may act as an inhibitor of GSK3b and thus may also participate in the
GSK3b-Axin-b-catenin complex as part of Wnt signaling pathway.
The discovery of GSKIP protein could also explain the conservation of this
protein throughout evolution rather than FRAT/GBP only in higher vertebrate.
Furthermore, in some extent, GSKIP or GSKIPtide as drug discovery of GSK3b
inhibitors in details need more exploration.