GSKIP, shares a homology of Axin GID domain and functions as a negative regulator of GSK3beta

1
GSKIP, shares a homology of Axin GID domain and
functions as a negative regulator of GSK3beta
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, Ching-Mei
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 Yat-Sen 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
4P-C-110
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-amino-acid 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.
Figure 5. GSKIP directly inhibits GSK3b activity.
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 GSKIP at various
doses (0, 0.2, 1, 5 µM). Arrow indicates
phosphorylated GST-Axin (275-510 aa), His-tagged
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).
Results
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 rhodamine-conjugated
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
(4145). b-catenin 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 Students t test
plt0.005, plt0.0005 versus control.
Figure 2. A 25-aa residue sequence of GSKIP is
high similarly to the GID of Axin. (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.
Figure 1. Protein sequence alignment of GSKIP and
its subcellular localization. (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.
• 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.

Figure 4. GSK3b phosphorylates GSKIP at S109 and
T113. (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.
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-Flag-GSK3b
and/or pcDNA-GSKIP or pcDNA-GSKIP(L130P) or pcDNA
vector. Immunoprecipitation (IP) was performed
with anti-Flag antibody. Western-blotting (WB)
was performed using anti-HA antibody. ?
indicates the GSKIP signal.