Inhibitory phosphorylation of glycogen synthase kinase-3 ... · 6/7/2003 · Lithium salts have...

Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3)in response to lithium: Evidence for autoregulation of GSK-3

Fang Zhang*, Christopher J. Phiel#, Laura Spece#, Nadia Gurvich*, and Peter S. Klein#,‡

*Department of Pharmacology, #Howard Hughes Medical Institute and Department of Medicine,(Division of Hematology-Oncology)

University of Pennsylvania School of Medicine415 Curie Blvd.

Philadelphia, PA 19104-6148

Running title: Autoregulation of GSK-3‡Author for correspondence: [email protected]

The abbreviations used are: GSK-3, glycogen synthase kinase-3; IMPase, inositolmonophosphatase; FRAT, frequently rearranged in advanced T-cell lymphoma; GBP, GSK-3binding protein; GID, GSK-3-interacting domain; TDZD-8, thiadiazolidinone-8; PDGF, platelet-derived growth factor; PKA, protein kinase A; p34cdc2/Cdk-1, cyclin-dependent kinase-1; p42MAPK/ERK2, mitogen-activated-protein kinase; siRNAs, short interfering RNAs; PP-1, proteinphosphatase-1; I-2, inhibitor-2; PKC, protein kinase C; BISIII, bisindoylmaleimide-III; RNAi,RNA interference; MEFs, mouse embryonic fibroblasts; AKAP220, A-kinase anchoringprotein220; PP-2A, protein phosphatase-2A; VPA, valproic acid.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 7, 2003 as Manuscript M212635200 by guest on A

ugust 27, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

2

Summary

Glycogen synthase kinase-3 (GSK-3) is a critical, negative regulator of diverse signaling

pathways. Lithium is a direct inhibitor of GSK-3, and has been widely used to test the putative

role of GSK-3 in multiple settings. However, lithium also inhibits other targets, including

inositol monophosphatase and structurally related phosphomonoesterases, and thus additional

approaches are needed to attribute a given biological effect of lithium to a specific target. For

example, lithium is known to increase the inhibitory amino (N)-terminal phosphorylation of

GSK-3, but the target of lithium responsible for this indirect regulation has not been identified.

We have characterized a short peptide derived from the GSK-3 interaction domain (GID) of

Axin that potently inhibits GSK-3 activity in vitro and in mammalian cells, and robustly

activates Wnt-dependent transcription, mimicking lithium action. We show here, using the GID

peptide as well as small molecule inhibitors of GSK-3, that lithium induces GSK-3 N-terminal

phosphorylation through direct inhibition of GSK-3 itself. Reduction of GSK-3 protein levels,

either by RNA interference or by disruption of the mouse GSK-3ß gene, causes increased N-

terminal phosphorylation of GSK-3, confirming that GSK-3 regulates its own phosphorylation

status. Finally, evidence is presented that N-terminal phosphorylation of GSK-3 can be

regulated by the GSK-3-dependent protein phosphatase-1/inhibitor-2 complex.

by guest on August 27, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3

Introduction

Lithium salts have provided effective pharmacotherapy for bipolar disorder for decades (1,2);

however, the underlying mechanisms of lithium action in psychiatric disorders are unknown (3-

5). Lithium also has diverse effects in other settings, including glycogen synthesis, embryonic

development, and synaptogenesis (4-6). Identification of the molecular target of lithium action

in these settings has been a focus of research over the past twenty years, and several potential

targets have been identified. For example, lithium inhibits glycogen synthase kinase-3 (GSK-3),

and this has been proposed to explain the effects of lithium on embryonic development and

glycogen synthesis (7). However, lithium also inhibits other targets, including inositol

monophosphatase (IMPase) (8), other phosphomonoesterases structurally related to IMPase

(5,9), phosphoglucomutase (10), and possibly other enzymes. For many of the known effects of

lithium, especially in the treatment of bipolar disorder, the relevant target(s) of lithium has not

been defined. Thus, in order to establish a prospective candidate as the target of lithium in a

given setting, new approaches that specifically interfere with the function of that candidate,

including development of more specific inhibitors and loss-of-function mutations in the genes of

interest, will be required in addition to demonstrating in vivo inhibition of a putative target by

lithium.

Inhibition of GSK-3 by lithium in vivo has been demonstrated in a number of systems, including

cultured cells, Xenopus oocytes and embryos, and adult rat brain (11-17). In many of these

studies, lithium activates pathways known to be inhibited by GSK-3. For example, GSK-3

inhibits glycogen synthase, and this is reversed by lithium (mimicking insulin action), most


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

4

likely through direct inhibition of GSK-3 (18). Similarly, GSK-3 antagonizes the canonical Wnt

signaling pathway, and lithium activates this pathway in cell culture and in intact embryos

(11,12,19,20). The argument that these effects of lithium are caused by inhibition of GSK-3,

rather than another target of lithium, is supported by loss-of-function mutations in GSK-3, which

are phenocopied by lithium in diverse organisms, including Dictyostelium and mouse (21,22).

In addition, numerous alternative inhibitors of GSK-3 have been described recently, and where

tested, these agents also mimic lithium effects on the Wnt pathway. Synthetic small molecule

inhibitors include hymenialdisine, paullones, indirubins and maleimide compounds, most of

which are ATP competitors (23-27). However, many of these compounds inhibit protein kinases

other than GSK-3. Polypeptide inhibitors of GSK-3 have also been described, including the

protein FRAT/GBP (frequently rearranged in advanced T-cell lymphoma/GSK-3 binding

protein) and peptides derived from the GSK-3ß-interacting domain (GID) of Axin (28,29).

FRAT/GBP inhibits GSK-3ß activity in vivo and is required for dorsal development in Xenopus

embryos (28). Axin is a scaffold protein that binds multiple components of the canonical Wnt

pathway, including GSK-3 and ß-catenin, to facilitate GSK-3-mediated phosphorylation and

degradation of ß-catenin (30,31). In contrast to the function of full-length Axin, fragments of

Axin that bind to GSK-3 (GID) inhibit GSK-3 activity in Xenopus oocytes (29). While the

mechanism of GSK-3 inhibition by FRAT/GBP or GID peptides is not known, these molecules

interact with GSK-3ß at a site remote from the ATP binding pocket, suggesting that the mode of

inhibition is distinct from the small molecule inhibitors described above (32,33).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

5

One intriguing consequence of lithium treatment is the increased phosphorylation of GSK-3

itself ((25,34-38) and data presented here). Several signaling pathways, including insulin,

PDGF, FGF, EGF, and reelin cause increased inhibitory phosphorylation of GSK-3 on either

serine-21 (in GSK-3α) or serine-9 (in GSK-3ß) (39). The ability of lithium to mimic this effect

in cerebellar granule cells was reported to be due to transient activation of Akt, but the direct

target of lithium responsible for this regulation was not identified (34). Lithium-induced

inhibitory phosphorylation of GSK-3 is especially interesting, as it provides additional, indirect

inhibition of GSK-3 that could reinforce or amplify the well-established direct inhibition of

GSK-3 by lithium.

We have addressed whether lithium-induced serine-21/9 phosphorylation arises through direct

inhibition of GSK-3 or through another lithium sensitive target using GID peptides, small

molecule GSK-3 inhibitors, and GSK-3 loss-of-function approaches. We have further

characterized GID peptides to show that they inhibit GSK-3 in multiple mammalian cell types

and that they are direct inhibitors of GSK-3 in vitro. We then show that increased serine-21/9

phosphorylation can be caused by GSK-3 inhibitors that function through several, distinct

mechanisms, and that reduction of GSK-3 expression by either RNA interference or GSK-3ß

knock out also results in increased serine-21/9 phosphorylation, providing strong support for the

autoregulation of GSK-3 activity. Finally, we propose that this autoregulation of GSK-3 is

mediated in part through GSK-3 regulation of the protein phosphatase-1/inhibitor-2 complex.

Experimental Procedures


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

6

Materials Lithium chloride (LiCl) was purchased from Sigma. Rottelerin, Quercetin,

Flavopiridol, Ro31-8220, TDZD-8, Rp-cAMPS, BISIII, LY 294002, staurosporine, and H-89

were purchased from Calbiochem and prepared in concentrated stock in either DMSO

(Rottelerin, Quercetin, Flavopiridol, Ro31-8220, TDZD-8, LY 294002, staurosporine, and H-89)

or sterile water (Rp-cAMPS and BISIII). Human recombinant Platelet-Derived Growth Factor-

BB (PDGF-BB, Sigma) was reconstituted in sterile 4 mM HCl containing 0.1% bovine albumin.

Human recombinant Insulin (Sigma) was dissolved in 0.01 N HCl.

Plasmids GID 320-429 (GID1-2, 109 aa) and GID380-404 (GID5-6, 25aa), full-length Axin and Axin

∆RGS in pCS2+MT vector were described previously (29). Leucine 390 to proline (L-P) mutants

were generated by site-directed mutagenesis of Xenopus axin based on the work of Smalley et al

(40). Tau plasmid T40/pSG5 was a gift of Dr. Virginia M.-Y. Lee (University of Pennsylvania,

Philadelphia, PA). pEGFP was purchased from CLONTECH. Luciferase constructs containing

three wild-type (Lef-OT) or three mutated (Lef-OF) Tcf/Lef binding sites were gifts from Dr.

Bert Vogelstein (John Hopkins University, Baltimore, MD) (41). Dominant negative Xtcf3

(dnTCF) lacks the N-terminal ß-catenin binding site (42). Inhibitor-2 was amplified from mouse

cDNA and cloned into pCS2+MT.

Cell Culture and Transfection 293T cells, Neuro2A and NIH3T3 cells were obtained from

American Type Culture Center and cultured as described elsewhere (43). Wild-type and GSK-3ß

knock-out mouse embryonic fibroblasts were gifts from Dr. James Woodgett (22). For

transfections, cells were plated in 6-well dishes at a density of 2.5 x 105 cells per well. Each

plasmid was transfected as follows: 1µg of Axin, ∆RGS, GID (or as dose-curve as indicated in


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

7

the figures) or empty vector control (either pCS2+ or pCS2+MT (myc-tag), indicated as pCS2

control in figures), 0.25µg of tau T40/pSG5; 0.2µg of Lef-OT or Lef-OF; 10ng of pRL-SV40

(Renilla luciferase); 0.5µg of dnTCF. All transfections included 0.3µg of pEGFP to assess

transfection efficiency (typically 50-80%). Fugene6 (Roche) was used for transfecting 293T or

Neuro2A cells. Firefly and Renilla luciferase activities were measured on a Monolight 3010

luminometer (Turner Designs) using the Dual Luciferase assay kit (Promega). For drug

treatment experiments, cells were serum-starved overnight before addition of drugs at

concentrations indicated in the figures, and harvested after indicated period of time.

Immunoblotting Cells were harvested at 24 hours after transfection or at indicated time points

for drug treatment using Reporter lysis buffer (Promega) or Passive lysis buffer (Promega) in the

case of Lef-OT experiments. Lysis buffer was supplemented with a mixture of protease and

phosphatase inhibitors (1:100, Sigma). Samples were centrifuged at 14,000 rpm for 10 min at

40C, and the supernatants were transferred to fresh tubes. Volume was adjusted to normalize

total protein concentration based on Bradford assays, and then Laemmli sample buffer was

added, followed by boiling for 5 min. Samples were separated by electrophoresis on 10%

polyacrylamide gels (SDS-PAGE), immunoblotted using PHF-1 antibody for p-tau (1:250,

provided by Peter Davie (44)), tau antibody T14/46(1:1000, provided by Dr.Virginia M.-Y. Lee),

GSK-3α/ß antibody (1:1000, Calbiochem), Akt1/PKBα antibody (1:1000, Sigma), myc antibody

(9E10, cell center of University of Pennsylvania) and hnRNP K (1:2000, provided by Dr. Gideon

Dryfress). Phospho-GSK-3ß (serine-9), phospho-GSK-3α/ß (serine-21/9), phospho-Akt (serine-

473) and phospho-p90 rsk (serine-381) (Cell Signaling), ß-catenin, and GSK-3ß antibodies


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

8

(Transduction Laboratories) were used at 1:1000 dilution. Immunoblots were visualized by

enhanced chemiluminescent detection (Amersham).

Immunofluorescent StainingNIH3T3 cells grown on cover-slips were co-transfected with 2µg

of GID380-404, GID(L->P)380-404, full-length Axin or pCS2+MT, together with 0.3µg of pEGFP,

using LipofectAMINE Plus. 24 hours after transfection, cells were fixed with chilled 4%

paraformaldehyde in PBS for 15 min, rinsed with PBS, permeabilized in 1% Triton-X100/10%

goat serum in PBS for 10 min, blocked in 0.1% Tween-20 /10% goat serum in PBS for 30 min,

incubated with ß-catenin antibody diluted 1:200 in blocking buffer for 60 min at RT, washed

with PBS, incubated with rhodamine-conjugated anti-mouse antibody (Jackson

ImmunoResearch) diluted 1:200 in blocking buffer for 45 min at RT, washed with PBS, then

mounted onto slides with VECTASHELD mounting media with DAPI (Vector Laboratories).

Cells were viewed at 40-fold magnification with a Leica DMR fluorescence microscope.

In vitro kinase assaysRecombinant GSK-3ß (500 units/ul) was purchased from New England

Bioloabs (NEB). GSK-3ß assay was performed as described previously (Klein 1996), except

that MgCl2 was 0.5 mM. GID320-429–his was affinity-purified by Ni-NTA (QIAGEN). GID380-404

and GID(L->P)380-404 peptides were chemically synthesized by the HHMI Structure Lab at the

University of California San Francisco and were purified by HPLC. FRATide was purchased

from Upstate Biotechnology. Recombinant ß-catenin protein purified from Sf9 cells was a

generous gift from Barry Gumbiner. Recombinant Inhibitor-2 was purchased from NEB and tau

was purified from bacteria as described (29). Prior to performing in vitro GSK-3 assays, we

tested each substrate at multiple concentrations and chose a concentration within the linear range


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

9

for the respective substrates. The final concentrations added to the reaction were as follows: ß-

catenin at 25µg/ml, tau at 12.5µg/ml, and I-2 at 25µg/ml. Tau protein was pre-phosphorylated

with MAP kinase as previously described (45). Reactions were stopped by adding Laemmli

sample buffer and boiling for 5 min. Samples were separated in 10% SDS-PAGE, followed by

Coomassie Blue staining. The gel was dried and scanned with STORM 840 PhosphoImager

(Molecular Dynamics). The intensity of the bands was quantified with Image Quant version 1.0

(Molecular Dynamics). The stained gel was scanned to confirm equal loading of GSK-3 and

substrates. PKA, p34cdc2 /Cdk-1, p42 MAPK/Erk2 and Casein Kinase II assays were performed as

previously described (46, Atherton-Fessler, 1993 #4414, Boulton, 1991 #1096, Russo, 1992

#4415). 20µM of H-89 was used for control as a PKA inhibitor, and 1µM of Staurosporine was

used for control as a p34cdc2 inhibitor.

siRNA Preparation and TransfectionShort interfering RNA oligonucleotides were synthesized

using the Gene Silencer kit from Ambion. Target sequences for hamster GSK-3α are: 5’ AAT

ATC GTG AGG CTG CGG TAC 3’, 5’ AAG GTG TAC ATG TAC CAG CTC 3’, 5’ AAG

GTG TGT GTC ACC GTG ACA 3’, 5’ AGC TGC TTC TCG GCC AGC CCA 3’, 5’ GCA CTT

TAC CAA GGC CAA GCT 3’. Target sequences for hamster GSK-3β are: 5’ AAC ATA GTC

CGA TTG CGG TAT 3’, 5’ AAG TTG TAT ATG TAT CAG CTG 3’, 5’ AAT TAG AGA AAT

GAA CCC AAA 3’, 5’ AGT TGT TGC TAG GAC AAC CAA 3’. One target sequence

recognizes both GSK-3 isoforms: 5’ CCT GGG GAC ACT GGT GTG GAT 3’. 25 nM of each

siRNA was transfected into CHO cells plated in 6-well dishes using Mirus Trans-IT TKO

transfection reagent and cells were harvested 48 hours later.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

10

PP-1 assay PP-1 assay was performed as previously described (47). Recombinant GSK-3ß

phosphorylated on serine-9 was used as substrate. I-2 (NEB) was added to a final concentration

of 1µM. ATP was added to a final concentration of 0.2mM. LiCl and GID380-404 were added at

the concentrations indicated in the figures. Serine-9 phosphorylation of GSK-3ß was detected by

WB with phospho-GSK-3ß (serine-9) antibody. GSK-3-mediated phosphorylation of I-2 was

detected by autoradiography by adding [γ-32P]ATP.

Results

A short GSK-3-binding peptide mimics lithium action in mammalian cells.

We have previously shown that a construct encoding 268aa derived from the GSK-3 interaction

domain (GID) of Axin (GID277-545) mimics lithium action in Xenopus embryos and that a

sequence as short as 25aa (GID380-404) inhibits overexpressed GSK-3ß in Xenopus oocytes (29).

To test whether GID peptides inhibit endogenous GSK-3 activity in mammalian cells, we have

transfected myc-tagged GID380-404 into mouse Neuro2A cells and assayed phosphorylation of the

microtubule-binding protein tau, a well-characterized substrate for GSK-3ß (44). Over-

expression of GID380-404 reduced tau phosphorylation, as determined by western blotting with a

phospho-tau specific antibody (PHF-1) and by the increased electrophoretic mobility of tau,

mimicking the effects of lithium (Fig 1A). The dominant-negative form of Axin, ∆RGS, also

inhibited tau phosphorylation in Neuro2A cells. Mutation of leucine390 to proline blocks

interaction of axin-GID with GSK-3 (40); GID380-404 containing this L->P mutation (GID(L-P)380-

404) failed to inhibit tau phosphorylation, indicating that GID must interact with GSK-3ß to


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

11

inhibit tau phosphorylation. In contrast, full-length Axin did not inhibit GSK-3ß-mediated tau

phosphorylation under these conditions (see also (29)).

Inhibition of GSK-3ß by Wnts or by lithium results in reduced phosphorylation and subsequent

accumulation of ß-catenin protein, and this has been widely employed as an indirect assay of

GSK-3ß activity (12,28,48,49). We therefore tested whether GID causes accumulation of ß-

catenin in Neuro2A cells. In contrast to full-length Axin, over-expression of GID320-429 or GID380-

404 caused accumulation of ß-catenin in a dose-dependent manner, mimicking the effect of lithium

(Fig 1B and C), and similar to observations in Xenopus oocytes (29). GID(L-P)320-429 or GID(L-

P)380-404 did not cause ß-catenin accumulation. GID-induced ß-catenin accumulation was evident

in both the cytoplasm and the nucleus, as visualized by immunofluorescence for ß–catenin in

NIH-3T3 cells (Fig 2A, B). Thus GID mimics lithium in causing ß-catenin accumulation in

mammalian cells.

As ß-catenin binds to TCF/Lef transcription factors to activate Wnt-responsive genes, we

evaluated the ability of GID to activate Wnt-dependent transcription using a TCF/Lef-luciferase

reporter (Lef-OT) (41). GID380-404 activated Lef-OT in a dose-dependent manner, mimicking

lithium, with a maximum activation over 200-fold (Fig. 2C, D). Lithium or GID-induced Lef-

OT activation was abolished by co-expression of a dominant-negative form of TCF (dnTCF) that

lacks the ß-catenin-binding site (42). Neither GID nor lithium activated the Lef-OF luciferase

reporter in which the TCF-binding sites are mutated (41). This suggests that activation of Lef-OT

by lithium and GID is mediated specifically by ß-catenin/TCF. These findings are similar to

observations showing activation of TOPFLASH by a larger fragment encoding GID derived


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

12

from mouse Axin (40). We have also found that a 23-residue GID peptide (aa382-404 of

Xenopus axin) retains GSK-3 binding and potently activates Lef-OT activity, while removal of 2

additional residues from either end markedly reduces activation of Lef-OT (data not shown).

Therefore short GID peptides mimic lithium in activating the canonical Wnt/ß-catenin signaling

pathway in mammalian cells.

GID inhibits GSK-3ß in vitro.

We previously observed that recombinant GID320-429 did not inhibit GSK-3 phosphorylation of a

pre-phosphorylated peptide derived from glycogen synthase (p-GS-2)(29). However recent

crystallographic and biochemical analyses of GSK-3ß have suggested that the activity of GSK-

3ß differs toward pre-phosphorylated (primed) and unphosphorylated (non-primed) substrates

(45,50-52). Indeed, a peptide derived from FRAT, FRATide, selectively inhibits GSK-3ß

phosphorylation of non-primed substrates (45). Therefore we have re-addressed the ability of

GID peptide to inhibit GSK-3 in vitro using alternative, non-primed substrates, including

recombinant ß-catenin (Fig 3A), tau protein (Fig 3B), and protein phosphatase-1 inhibitor-2 (data

not shown). When recombinant GID320-429 purified as a his-tagged peptide or GID380-404 as a

chemically-synthesized peptide was added to in vitro GSK-3ß assays, phosphorylation of each of

these non-primed substrates was inhibited (Fig 3 A, B and data not shown). GID peptides

inhibited GSK-3ß at approximately 1:1 stoichiometry with GSK-3ß. As reported for p-GS-2,

GID did not inhibit GSK-3ß-mediated phosphorylation of p-CREB (data not shown), a widely

used primed GSK-3 substrate derived from CREB (53,54).


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

13

Thus inhibition of GSK-3ß by GID peptides appears to depend on the substrate, but from the

data presented so far, it is not clear whether this is related to substrate sequence or the presence

of a priming phosphate. Therefore tau protein pre-phosphorylated with MAP kinase was

compared with non-primed tau in the in vitro GSK-3ß assay. As reported previously (45,55), pre-

phosphorylation of tau protein by MAP Kinase facilitated GSK-3ß-mediated phosphorylation of

tau (Fig 3B, compare lane 8 with lane 3). Inhibition of GSK-3ß by GID was significantly less

efficient when pre-phosphorylated tau was used as substrate (Fig 3B, compare lane 11 with lane

6). However, a five-fold higher concentration of GID inhibited phosphorylation of both non-

primed and primed tau. This suggests that GID preferentially inhibits GSK-3ß activity toward

non-primed substrates, but can also inhibit phosphorylation of primed substrates at higher

concentrations.

To determine whether GID inhibits other serine/threonine protein kinases, we assayed Protein

Kinase A (PKA), p34cdc2 / Cdk-1, Casein Kinase II, and MAP Kinase in vitro in the presence of

GID320-429-his or GID380-404 peptide. PKA-mediated phosphorylation of kemptide was not

inhibited by GID even at high concentrations, but was effectively inhibited by the PKA inhibitor

H-89 at 20µM (Fig 3C)(56,57). Similarly, p34cdc2 phosphorylation of Histone H1 was not

inhibited by GID but was inhibited by staurosporine at 1µM (24). GID also did not inhibit the in

vitro activity of Casein Kinase II or MAP Kinase (data not shown). Therefore GID is not a

general inhibitor of serine/threonine protein kinases.

GBP/FRAT binds and also inhibits GSK-3, and the affinity of FRATide (a 39aa peptide

corresponding to residues 188-226 of FRAT1) for GSK-3 is reportedly 100-fold higher than


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

14

GID380-404 peptide (58). However, the potency of GID and FRAT in inhibiting GSK-3 was not

compared in that study. Thus, we transfected 293T cells with plasmids encoding myc-GID380-404

or myc-GBP at varying concentrations together with tau and Lef-OT reporter. We found that

GID380-404 inhibited tau phosphorylation more potently than GBP, as assessed by western blot

with PHF-I antibody (Fig 4A, top panel) and electrophoretic mobility shift (Fig 4A, second

panel). GID380-404 also activated Lef-OT reporter more robustly than GBP (Fig 4B). In vitro, the

IC50 for GID380-404 inhibition of GSK-3ß was ~50nM and the IC50 of FRATide was ~160nM (Fig

4C). Therefore, despite the apparently lower affinity of GID380-404 peptide for GSK-3, GID380-404

and GID320-429 are potent GSK-3ß inhibitors.

GSK-3 inhibitors cause increased phosphorylation of GSK-3.

Although lithium is a direct inhibitor of GSK-3, in vivo it also causes increased phosphorylation

of GSK-3ß on serine-9 (25,34-37), which inhibits GSK-3ß activity (39,59-61). Various

mechanisms have been proposed to explain this observation, including activation of Akt/PKB

(34,62). We have also observed robust and rapid (within 5 min) serine-9 phosphorylation after

treatment with lithium in 293T, Neuro2A, and NIH3T3 cells (Figs 5 and 8). This effect of

lithium could be mediated through inhibition of GSK-3 or another target of lithium. Therefore

we tested whether alternative inhibitors of GSK-3 also cause increased phosphorylation of GSK-

3ßser9. We reasoned that if GSK-3ßser9 phosphorylation is increased by treatment with diverse

agents that inhibit GSK-3 through distinct mechanisms, it is likely that this response is mediated

through inhibition of GSK-3 per se. We found that, similar to lithium treatment, GID expression

caused increased GSK-3ßser9 phosphorylation in a dose-dependent manner in both 293T cells (Fig

5C) and Neuro2A cells (data not shown). This is in contrast to full length Axin, which reduced


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

15

GSK-3ßser9 phosphorylation (Fig 5B; (63)). GBP expression also caused increased

phosphorylation of GSK-3ßser9 (data not shown).

The observation that both GID and lithium increase GSK-3ßser9 phosphorylation suggests that this

effect is mediated through inhibition of GSK-3. To test this further, we used a variety of small

molecule inhibitors of GSK-3, including both ATP-competitive inhibitors (such as Flavoperidol,

Rottlerin, and Quercetin) and TDZD, which reportedly does not interact with the ATP binding

pocket (24,27,56). Flavopiridol, Rottlerin, Quercetin, and TDZD all caused increased GSK-3ßser9

phosphorylation in Neuro2A cells at concentrations that inhibit GSK-3ß (Fig 6A, C), and

increased phosphorylation of GSK-3α and ß caused by Rottlerin at 1µM or TDZD at 50µM was

apparent within 5 min., similar to lithium treatment (Fig 6B, D). Therefore GSK-3 inhibitors

acting through distinct mechanisms cause increased N-terminal phosphorylation of GSK-3. In

contrast, Ro31-8220, a PKC inhibitor that also inhibits GSK-3 activity (64), blocked

phosphorylation of GSK-3 (Fig 6A, lanes 11-12 and Fig 6B, lanes 8-11), similar to the results

reported for the GlaxoSmithKline GSK-3 inhibitors SB-216763 and SB-415286, maleimides

structurally similar to Ro 31-8220 (25). However, Ro31-8220, SB-216763, and SB-415286 also

inhibit p90rsk2 (56,65), which phosphorylates GSK-3ßser9 in vitro (66,67), suggesting that p90rsk2 or

a related kinase may phosphorylate GSK-3 in vivo as well. Indeed, bisindoylmaleimide-III (BIS-

III), which potently inhibits p90rsk2 but only weakly inhibits GSK-3 (56), blocks both basal and

lithium-induced serine-9 phosphorylation (Fig 8F, lanes 2 and 5). Valproic acid (VPA), which is

also used to treat bipolar disorder and affects neuronal growth cones in a manner similar to

lithium (36,68), does not inhibit GSK-3 in our assays (43) and does not cause increased


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

16

phosphorylation of GSK-3ß under these conditions (Fig 6C), as described previously (36),

although VPA does cause increased serine-9 phoshorylation in other settings (38).

Reduced expression of GSK-3 leads to increased N-terminal phosphorylation

Since a number of GSK-3 inhibitors acting through at least three distinct mechanisms similarly

increased GSK-3α/ß phosphorylation, the simplest explanation is that this phosphorylation is

regulated by GSK-3 itself. However, the formal possibility remains that each of these inhibitors

affects target(s) other than GSK-3 that all regulate GSK-3 phosphorylation in some manner.

Therefore we tested the hypothesis that GSK-3 negatively regulates its own phosphorylation by

reducing GSK-3 expression using RNA interference (RNAi) (69,70) or disruption of the mouse

GSK-3ß locus (22). Using short interfering RNAs (siRNAs) targeted specifically against GSK-

3α or GSK-3ß, we reduced the protein level of GSK-3α or ß respectively (Fig 7A). When GSK-

3α was depleted, phosphorylation of GSK-3ßser9 increased without a change in GSK-3ß protein

level (Fig 7A, lane 3). (In addition, the amount of phosphorylated GSK-3α did not change

despite the reduced level of GSK-3α protein, indicating a relative increase in GSK-3α

phosphorylation). Conversely, when GSK-3ß was depleted (Fig 7A, lane 4), phosphorylation of

GSK-3αser21 increased (the relative phosphorylation of GSK-3ßser9 also increased, when

normalized to overall protein levels). Therefore, depletion of either isoform of GSK-3 increases

N-terminal phosphorylation. Alternatively, GSK-3ß expression is completely absent in

embryonic fibroblasts (MEFs) derived from mice in which the GSK-3ß gene has been disrupted,

but these cells still express GSK-3α (Fig 7B)(22). In these GSK-3ß-/- MEFs, serine-21

phosphorylation of GSK-3α is increased compared to GSK-3α in wild-type MEFs (Fig 7B,

GSK-3ß+/+ , lane 1 vs. GSK-3ß-/- lane 4). Therefore, reduction of GSK-3 activity either by RNAi


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

17

or by gene disruption of a specific GSK-3 isoform leads to increased N-terminal phosphorylation

of the complementary isoform. Furthermore, overexpression of the constitutively active GSK-

3ßS9A mutant causes decreased serine-9 phosphorylation of endogenous GSK-3ß (71). Taken

together with the effects of GSK-3 inhibitors such as GID and lithium, these data strongly

support a role for GSK-3 in the autoregulation of serine-21/9 phosphorylation.

Autoregulation of GSK-3 phosphorylation via GSK-3-activated protein phosphatase-1.

GSK-3 could regulate serine-21/9 phosphorylation indirectly by inhibiting a serine/threonine

kinase as shown in the diagram in Fig 8A, or by activating a phosphatase (Fig 9A). Akt, p90rsk2,

PKA, and p70S6K are known to phosphorylate serine-9 in vitro (57,59,66,72,73). Consistent with

previously reported data in cerebellar granule cells (34), lithium caused increased

phosphorylation of Akt on serine 473 (associated with activation of Akt) in Neuro2A cells (Fig

8B). However, this lithium-induced Akt phosphorylation/activation appears to be cell-type-

specific. In NIH3T3 (Fig 8C), 293T (Fig 8D), and mouse embryonic fibroblasts (data not

shown), lithium treatment does not affect Aktser473 phosphorylation at anytime between 5min and

24 hours, while the increase in GSK-3 phosphorylation occurs within 5 minutes of lithium

treatment (Fig 8C, D). This suggests that lithium-induced serine-9 phosphorylation does not

require activation of Akt. Furthermore, pretreatment with LY-294002, an inhibitor of

phosphatidylinositol-3 kinase (PI3K) (74), did not block GSK-3 phosphorylation caused by

lithium in Neuro2A cells (Fig 8B, lane 7) or in NIH3T3 cells (Fig 8C, lane 4, 7 and 9),

supporting that the increase in GSK-3 phosphorylation caused by lithium is largely independent

of PI-3K and Akt. To test whether GSK-3 inhibitors cause increased GSK-3ser9 phosphorylation

by activating p90rsk2, we compared the time-course of p90rsk2 activation with GSK-3ßser9


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

18

phosphorylation after lithium or Rottlerin treatment. While both lithium and Rottlerin activated

p90rsk, as detected by an antibody recognizing the phospho-p90rsk1/2, the time course was

significantly delayed relative to phosphorylation of GSK-3ßser9 (Fig 8E). This suggests that

although p90rsk2 may be involved in phosphorylation of serine-9 in vivo, activation of p90rsk2 is

not sufficient to explain the rapid increase in phospho-GSK-3ßser9 caused by GSK-3ß inhibitors.

Pretreatment with a PKA inhibitor (Rp-cAMPS) (75) or a p70S6K inhibitor (rapamycin) (76) has

no effect on either endogenous or lithium-induced GSK-3ßser9 phosphorylation (Fig 8F, lane 4

and data not shown), suggesting that lithium-induced GSK-3 phosphorylation is not mediated

through activation of PKA or p70S6K.

Alternatively, GSK-3 inhibitors could block GSK-3-mediated activation of a phosphatase (Fig

9A). As GSK-3 activates protein phosphatase-1 (PP-1) by phosphorylation of Inhibitor-2 (I-2),

an inhibitory subunit of the PP-1 complex (77-82), lithium, GID, and other GSK-3 inhibitors

may prevent phosphorylation of I-2, leading to inhibition of PP-1 by I-2 and thus reduced

dephosphorylation of GSK-3 (Fig 9A). To test this hypothesis, we first investigated whether

GSK-3ßser9-P can be dephosphorylated by PP-1. In an in vitro phosphatase assay, PP-1α

significantly reduced GSK-3ßser9 phosphorylation (Fig 9B, lane 2, top panel). The

dephosphorylation of serine-9 by PP-1 was abolished by addition of I-2 (Fig 9B, lane 3, top

panel). However, when GSK-3ß was activated by addition of ATP, serine-9 phosphorylation of

GSK-3ß was reduced (Fig 9B, lane 4, top panel), presumably due to activation of PP-1 through

GSK-3ß-mediated phosphorylation and inhibition of I-2. Interestingly, addition of lithium, GID

(Fig 9B, lane 5 and 6, top panel) or SB-216763 (not shown) all prevented dephosphorylation of

serine-9, supporting the hypothesis that GSK-3 inhibitors block GSK-3-mediated activation of


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

19

PP-1 by suppressing GSK-3 phosphorylation of I-2. To exclude the possibility that these GSK-3

inhibitors directly inhibit PP-1, we added lithium or GID to the PP-1 reaction in the absence of I-

2; neither lithium nor GID affected PP-1 activity toward phosphorylated GSK-3ß (Fig 9C, lane 3

and 4, top panel) or kemptide (data not shown).

Supporting these in vitro observations, overexpression of mouse I-2 in 293T cells (Fig 9D) or

Neuro2A cells (data not shown) causes increased phosphorylation of GSK-3αser21 and ßser9,

suggesting that PP-1 can regulate GSK-3 phosphorylation in vivo. Furthermore, GSK-3ß activity

has also been proposed to be regulated by PP-1, based on the effects of phosphatase inhibitors in

cultured cells and brain slices (83,84). Taken together, these results suggest a mechanism by

which GSK-3, through phosphorylation and inhibition of I-2, activates PP-1 to reduce serine-

21/9 phosphorylation, and that increased serine-21/9 phosphorylation by lithium or GID is a

consequence of inhibition of GSK-3.

Discussion

Lithium inhibits GSK-3 in vitro and in vivo, and this inhibition may explain many of the

effects of lithium; however, lithium also inhibits, at similar or lower concentrations,

phosphoglucomutase, a family of phosphomonoesterases that includes inositol

monophosphatase, and possibly other targets yet to be identified. Because lithium salts

are widely used therapeutically as well as experimentally in diverse settings, it is

important to develop approaches to distinguish which, if any, of these direct targets is

responsible for a given effect of lithium. Several groups have recently reported that


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

20

lithium causes increased phosphorylation of GSK-3ß on serine-9, but the direct target of

lithium responsible for this effect has not been determined. We have therefore used

pharmacological and genetic approaches to test whether the target of lithium in this

setting is GSK-3. We find that inhibition of GSK-3 by lithium, by peptide or small

molecule inhibitors, or through reduction in GSK-3 expression leads to increased N-

terminal phosphorylation of GSK-3α and ß, demonstrating autoregulation of GSK-3 N-

terminal serine phosphorylation.

One approach to validating a proposed target of lithium is to test whether alternative

inhibitors of the putative target mimic the effect of lithium. To this end, we have

developed a short peptide inhibitor of GSK-3 derived from the GSK-3 interaction domain

of axin, which we have termed GID. We previously showed that GID peptides as short

as 25 amino acids inhibit overexpressed GSK-3ß in Xenopus oocytes. Further

characterization here shows that GID potently inhibits endogenous GSK-3 activity in

mammalian cells, as measured by reduced phosphorylation of tau protein, accumulation

of ß-catenin protein, and robust (over 200-fold) activation of ß-catenin/Tcf-dependent

transcription, mimicking lithium-mediated inhibition of GSK-3. Activation of a Wnt

dependent transcription reporter has also been reported by others using a larger fragment

of Axin containing the GID (40); while this effect was interpreted as a consequence of

disruption of the axin complex, our data show that GID peptides directly inhibit GSK-3

activity in vivo and in vitro (these distinct mechanisms are not mutually exclusive). In

this respect, GID peptides are similar to FRAT/GBP, which also inhibits GSK-3 activity

in Xenopus oocytes, mammalian cells, and in in vitro assays (28,45,58). We had


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

21

previously reported (29) that a larger fragment of axin containing the GID did not inhibit

in vitro GSK-3ß phosphorylation of a prephosphorylated peptide derived from glycogen

synthase (GS-2). However, new insights into the structure of GSK-3 have suggested that

the mechanism of phosphorylation may differ between prephosphorylated and

unphosphorylated substrates, and prior observations with FRAT showed that, in vitro, a

FRAT peptide inhibits GSK-3ß phosphorylation of unphosphorylated, but not

prephosphorylated tau protein (45). We therefore re-examined GID inhibition of GSK-3ß

in vitro and found that GID more potently inhibits GSK-3 activity toward

unphosphorylated substrates, indicating an unexpected substrate preference for inhibition.

In contrast to our findings that full length axin does not inhibit GSK-3 in vivo when

expressed at levels similar to GID or ∆RGS (this work and (29)), a recent paper reports

that overexpression of full length axin can inhibit GSK-3 mediated phosphorylation of

tau (85). While the level of axin overexpression was not indicated in that work, we also

find that, at considerably higher levels of expression, full length axin can inhibit GSK-3

overexpressed in Xenopus oocytes (data not shown), consistent with those reported

findings. The mechanism of this inhibition is currently being investigated.

We have also found that several of the small molecule inhibitors of GSK-3, like lithium,

cause rapid (within 5 minutes) increased N-terminal serine phosphorylation of GSK-3α

and ß. Most of these inhibitors appear to function by occluding the ATP binding pocket,

and in many cases, these inhibitors have been shown to inhibit other protein kinases.

GID and FRAT/GBP interact with residues on the GSK-3 dimer interface, far from the

ATP binding pocket, suggesting that these polypeptides inhibit GSK-3 through a distinct


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

22

mechanism (32,33). A crystal structure of GSK-3ß bound to a 19-residue peptide from

the axin-GID has been described recently that confirms this site of interaction with GSK-

3ß (86); this 19-mer did not cause a significant change in the conformation of the GSK-

3ß active site; however, in our assays, a similar peptide also did not inhibit GSK-3ß

activity (data not shown). Furthermore, TDZD, a novel GSK-3 inhibitor that reportedly

does not compete with ATP, also causes increased serine-21/9 phosphorylation. Thus,

increased serine-21/9 phosphorylation can be caused by agents that inhibit GSK-3

through several, distinct mechanisms (lithium, GID, TDZD, and ATP site competitors).

A few of the ATP competitors, including Ro31-8220, SB-216763 and SB-415286, inhibit

GSK-3 but do not cause increase serine-21/9 phosphorylation (figure 6A and (23)).

However, these compounds also inhibit p90rsk2, and we find that inhibition of p90rsk2, even

with inhibitors that do not inhibit GSK-3 (e.g. BIS-III, see Fig 8F), reduces basal and

lithium stimulated serine-21/9 phosphorylation.

While the likeliest explanation for the parallel effect of diverse GSK-3 inhibitors on

GSK-3 phosphorylation is that they act through inhibition of GSK-3, the possibility

remains that each of these agents interacts with a cryptic target that independently

regulates GSK-3 phosphorylation. To test further whether GSK-3 regulates its own

phosphorylation, we have used two loss-of-function approaches: RNA interference in

CHO cells and disruption of the GSK-3ß gene, using a mouse embryonic fibroblast

(MEF) cell line derived from the GSK-3ß knock out mouse (22). When GSK-3α levels

are reduced by RNAi, phosphorylation of GSK-3ßser9 increases markedly without a

change in overall level of GSK-3ß (the relative phosphorylation of GSK-3αser21 also


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

23

increased). Similarly, when GSK-3ß levels are reduced, either by RNAi or in GSK-3ß

deficient MEFs, phosphorylation of GSK-3αser21 is increased. Furthermore,

overexpression of GSK-3ßS9->A, which raises GSK-3 activity without changing the level

of GSK-3ser9, decreases phosphorylation of endogenous GSK-3ß on serine-9 (71). Taken

together with the inhibitor data presented here, these observations demonstrate

autoregulation of GSK-3α and ß and strongly support the argument that the effect of

lithium on N-terminal phosphorylation is mediated by inhibition of GSK-3.

GSK-3 autoregulation could involve inhibition of a kinase or activation of a phosphatase.

Protein kinases known to phosphorylate GSK-3α/ß at serine-21/9 include Akt, p90rsk2,

PKA, and p70S6K. Modest activation of Akt by lithium in cerebellar granule cells has been

reported previously, and we find similar modestly increased phosphorylation of Akt,

indicative of activation, in Neuro 2A cells, supporting a role for GSK-3 in the regulation

of Akt activation (87). However, we also observe lithium-induced serine-21/9

phosphorylation that is independent of Akt activation. In fibroblasts, where basal Akt

phosphorylation and activity are low, or in 293T cells, where basal Akt activity and

phosphorylation are high, lithium increases serine-21/9 phosphorylation without

changing the level of Akt phosphorylation, and this increased GSK-3 phosphorylation is

not blocked by inhibition of PI-3 kinase, indicating that the effect does not require

activation of Akt. Furthermore, activation of p90rsk2 is also not sufficient to explain

lithium-induced phosphorylation of GSK-3α/ß, as p90rsk activation is significantly slower

than the phosphorylation of GSK-3α/ß following addition of lithium (although p90rsk may

contribute to basal GSK-3 phosphorylation). Inhibition of PKA (with Rp- cAMPS) or


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

24

p70S6K (with rapamycin) does not block endogenous or lithium-induced GSK-3

phosphorylation, suggesting that lithium-induced phosphorylation of GSK-3α/ß is not

mediated through activation of PKA or p70S6K. While these findings cannot rule out

regulation of another serine-21/9 kinase (such as protein kinase C or integrin linked

kinase, see (88)), none of the four protein kinases that we tested appears to explain the

autoregulation of GSK-3α/ß, and we turned to GSK-3-mediated activation of a

phosphatase as an alternative mode of regulation.

It is well-documented that GSK-3 activates PP-1 by phosphorylating inhibitor-2 (I-2), an

inhibitory subunit of the PP-1 complex (77-82). Furthermore, PP-1 and GSK-3, as well

as PKA, interact with the AKAP220 protein and, based on the effect of phosphatase

inhibitors in cultured cells or rat brain slices, PP-1 has been proposed to activate GSK-3

(83,84), although PP-1 regulation of GSK-3 N-terminal phosphorylation was not

examined in those studies. If PP-1 dephosphorylates serine-21/9, then this would be

inhibited by I-2. Increased GSK-3 activity would then enhance phosphorylation and

inhibition of I-2, leading to activation of PP-1 and thus diminished serine-21/9

phosphorylation, while inhibition of GSK-3 would allow inhibition of PP-1 by I-2, with

consequent increased serine-21/9 phosphorylation. We demonstrate this by showing that

GSK-3ßser9P can be dephosphorylated by PP-1, and that, in the absence of ATP, this is

inhibited by I-2. Importantly, addition of ATP activates GSK-3 allowing

phosphorylation of I-2 and reactivation of PP-1, resulting in GSK-3ßser9P

dephosphorylation. GSK-3 inhibitors (lithium or GID) block phosphorylation of I-2,

leading to suppression of PP-1 and thus serine-9 phosphorylation is maintained.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

25

Therefore, GSK-3 can regulate its own phosphorylation status through regulation of the

PP-1/I-2 complex.

Protein phosphatase 2A (PP-2A) is also able to dephosphorylate GSK-3ß in vitro (60,66),

and has been proposed to regulate GSK-3ß activity and N-terminal phosphorylation in

vivo (37,89). Furthermore, lithium can block ceramide-induced activation of a protein

phosphatase with characteristics similar to PP-2A (89). This finding could imply that

GSK-3 regulates PP-2A in a manner similar to the regulation of PP-1. While GSK-3

regulation of PP-2A has not been described, our data do not exclude this possibility.

Nevertheless, our findings taken together with previous work (83,84) strongly support a

role for PP-1 in the autoregulation of GSK-3 N-terminal

phosphorylation/dephosphorylation.

In most settings, GSK-3 is a constitutively active protein kinase that is inhibited in

response to extracellular signals. Regulation of GSK-3 N-terminal phosphorylation by

GSK-3 itself confers an unusual feedback system that could help to maintain GSK-3 in an

active state but could also amplify an inhibitory signal and accelerate the conversion of

GSK-3 to an inactive state. In the response to lithium, this autoregulation implies two

levels of inhibition: rapid, direct inhibition of GSK-3 by lithium followed by inactivation

of PP-1 and consequent increased inhibitory phosphorylation of GSK-3. Since GSK-3

function in the Wnt pathway appears to be insulated from the effects of inhibitory, N-

terminal kinases such as Akt (63), we predict that this increased inhibitory

phosphorylation of GSK-3 would primarily affect Wnt-independent GSK-3 regulated


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

26

pathways. This suggests an unexpected level of signaling pathway selectivity in lithium

action that warrants further investigation.

References

1. Cade, J. (1949) Med. J. Aust. 2, 349-352

2. Rogers, C. (1989) Aust Nurses J 19(2), 29

3. Manji, H. K., and Lenox, R. H. (1999) Biol Psychiatry 46(10), 1328-1351

4. Williams, R. S., and Harwood, A. J. (2000) Trends Pharmacol Sci 21(2), 61-4

5. Phiel, C. J., and Klein, P. S. (2001) Annu Rev Pharmacol Toxicol 41, 789-813

6. Salinas, P. C. (1999) Biochem Soc Symp 65, 101-109

7. Klein, P. S., and Melton, D. A. (1996) Proc. Nat'l. Acad. Sci. U.S.A. 93, 8455-8459

8. Berridge, M. J., Downes, C. P., and Hanley, M. R. (1989) Cell 59(3), 411-419

9. York, J. D., Ponder, J. W., and Majerus, P. W. (1995) Proc. Natl. Acad. Sci. 92, 5149-

5153

10. Ray, W. J., Jr., Szymanki, E. S., and Ng, L. (1978) Biochim Biophys Acta 522(2), 434-42.

11. Stambolic, V., Ruel, L., and Woodgett, J. (1996) Curr. Biol. 6, 1664-1668

12. Hedgepeth, C., Conrad, L., Zhang, Z., Huang, H., Lee, V., and Klein, P. (1997) Devel.

Biol. 185, 82-91

13. Hong, M., Chen, D. C., Klein, P. S., and Lee, V. M.-Y. (1997) J. Biol. Chem. 272(40),

25326-25332

14. Munoz-Montano, J. R., Moreno, F. J., Avila, J., and Diaz-Nido, J. (1997) FEBS Lett

411(2-3), 183-188


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

27

15. Lovestone, S., Davis, D. R., Webster, M. T., Kaech, S., Brion, J. P., Matus, A., and

Anderton, B. H. (1999) Biol Psychiatry 45(8), 995-1003

16. Takahashi, M., Yasutake, K., and Tomizawa, K. (1999) J Neurochem 73(5), 2073-83

17. Lucas, F. R., Goold, R. G., Gordon-Weeks, P. R., and Salinas, P. C. (1998) J Cell Sci

111(Pt 10), 1351-1361

18. Summers, S. A., Kao, A. W., Kohn, A. D., Backus, G. S., Roth, R. A., Pessin, J. E., and

Birnbaum, M. J. (1999) J Biol Chem 274(25), 17934-40

19. Schneider, S., Steinbeisser, H., Warga, R. M., and Hausen, P. (1996) Mech Dev 57(2),

191-198

20. Larabell, C. A., Torres, M., Rowning, B. A., Yost, C., Miller, J. R., Wu, M., Kimelman,

D., and Moon, R. T. (1997) J Cell Biol 136(5), 1123-1136

21. Harwood, A. J., Plyte, S. E., Woodgett, J., Strutt, H., and Kay, R. R. (1995) Cell 80(1),

139-148

22. Hoeflich, K. P., Luo, J., Rubie, E. A., Tsao, M. S., Jin, O., and Woodgett, J. R. (2000)

Nature 406(6791), 86-90

23. Coghlan, M. P., Culbert, A. A., Cross, D. A., Corcoran, S. L., Yates, J. W., Pearce, N. J.,

Rausch, O. L., Murphy, G. J., Carter, P. S., Roxbee Cox, L., Mills, D., Brown, M. J.,

Haigh, D., Ward, R. W., Smith, D. G., Murray, K. J., Reith, A. D., and Holder, J. C.

(2000) Chem Biol 7(10), 793-803.

24. Leclerc, S., Garnier, M., Hoessel, R., Marko, D., Bibb, J. A., Snyder, G. L., Greengard,

P., Biernat, J., Wu, Y. Z., Mandelkow, E. M., Eisenbrand, G., and Meijer, L. (2001) J

Biol Chem 276(1), 251-260


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

28

25. Lochhead, P. A., Coghlan, M., Rice, S. Q., and Sutherland, C. (2001) Diabetes 50(5),

937-46.

26. Martinez, A., Alonso, M., Castro, A., Perez, C., and Moreno, F. J. (2002) J Med Chem

45(6), 1292-9.

27. Martinez, A., Castro, A., Dorronsoro, I., and Alonso, M. (2002) Med Res Rev 22(4), 373-

84.

28. Yost, C., Farr, G. H. r., Pierce, S. B., Ferkey, D. M., Chen, M. M., and Kimelman, D.

(1998) Cell 93(6), 1031-1041

29. Hedgepeth, C. M., Deardorff, M. A., Rankin, K., and Klein, P. S. (1999) Mol Cell Biol

19(10), 7147-7157

30. Zeng, L., Fagotto, F., Zhang, T., Hsu, W., Vasicek, T. J., Perry, W. L., Lee, J. J.,

Tilghman, S. M., Gumbiner, B. M., and Costantini, F. (1997) Cell 90, 181-192

31. Kikuchi, A. (1999) Cell Signal 11(11), 777-788

32. Fraser, E., Young, N., Dajani, R., Franca-Koh, J., Ryves, J., Williams, R. S., Yeo, M.,

Webster, M. T., Richardson, C., Smalley, M. J., Pearl, L. H., Harwood, A., and Dale, T.

C. (2002) J Biol Chem 277(3), 2176-85.

33. Bax, B., Carter, P. S., Lewis, C., Guy, A. R., Bridges, A., Tanner, R., Pettman, G.,

Mannix, C., Culbert, A. A., Brown, M. J., Smith, D. G., and Reith, A. D. (2001) Structure

(Camb) 9(12), 1143-52.

34. Chalecka-Franaszek, E., and Chuang, D. M. (1999) Proc Natl Acad Sci U S A 96(15),

8745-50.

35. Bhat, R. V., Shanley, J., Correll, M. P., Fieles, W. E., Keith, R. A., Scott, C. W., and Lee,

C. M. (2000) Proc Natl Acad Sci U S A 97(20), 11074-9.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

29

36. Hall, A. C., Brennan, A., Goold, R. G., Cleverley, K., Lucas, F. R., Gordon-Weeks, P. R.,

and Salinas, P. C. (2002) Mol Cell Neurosci 20(2), 257-70.

37. Song, L., De Sarno, P., and Jope, R. S. (2002) J Biol Chem 277(47), 44701-8.

38. De Sarno, P., Li, X., and Jope, R. S. (2002) Neuropharmacology 43(7), 1158-1164

39. Manoukian, A. S., and Woodgett, J. R. (2002) Adv Cancer Res 84, 203-29

40. Smalley, M. J., Sara, E., Paterson, H., Naylor, S., Cook, D., Jayatilake, H., Fryer, L. G.,

Hutchinson, L., Fry, M. J., and Dale, T. C. (1999) Embo J 18(10), 2823-2835

41. Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger, R., Kinzler, K. W.,

Vogelstein, B., and Clevers, H. (1997) Science 275, 1784-1787

42. Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S.,

Korinek, V., Roose, J., Destree, O., and Clevers, H. (1996) Cell 86, 391-399

43. Phiel, C. J., Zhang, F., Huang, E. Y., Guenther, M. G., Lazar, M. A., and Klein, P. S.

(2001) Journal of Biological Chemistry 276(39), 36734-41

44. Greenberg, S. G., Davies, P., Schein, J. D., and Binder, L. I. (1992) J. Biol. Chem. 267,

564-569

45. Thomas, G. M., Frame, S., Goedert, M., Nathke, I., Polakis, P., and Cohen, P. (1999)

FEBS Lett 458(2), 247-51.

46. Slice, L. W., and Taylor, S. S. (1989) J Biol Chem 264(35), 20940-6.

47. MacKintosh, C., Garton, A. J., McDonnell, A., Barford, D., Cohen, P. T., Tonks, N. K.,

and Cohen, P. (1996) FEBS Lett 397(2-3), 235-8.

48. Miller, J. R., and Moon, R. T. (1996) Genes and Development 10, 2527-2539

49. Yost, C., Torres, M., Miller, J., Huang, E., Kimelman, D., and Moon, R. (1996) Genes &

Development 10, 1443-1454


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

30

50. Dajani, R., Fraser, E., Roe, S. M., Young, N., Good, V., Dale, T. C., and Pearl, L. H.

(2001) Cell 105(6), 721-32.

51. Frame, S., Cohen, P., and Biondi, R. M. (2001) Mol Cell 7(6), 1321-7.

52. ter Haar, E., Coll, J. T., Austen, D. A., Hsiao, H. M., Swenson, L., and Jain, J. (2001) Nat

Struct Biol 8(7), 593-6.

53. Wang, Q. M., Fiol, C. J., DePaoli-Roach, A. A., and Roach, P. J. (1994) J Biol Chem

269(20), 14566-14574

54. Chen, G., Huang, L. D., Jiang, Y. M., and Manji, H. K. (1999) J Neurochem 72, 1327-

1330

55. Goedert, M., Cohen, E. S., Jakes, R., and Cohen, P. (1992) FEBS Lett 312(1), 95-9.

56. Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Biochem J 351(Pt 1), 95-

105.

57. Li, M., Wang, X., Meintzer, M. K., Laessig, T., Birnbaum, M. J., and Heidenreich, K. A.

(2000) Mol Cell Biol 20(24), 9356-63.

58. Culbert, A. A., Brown, M. J., Frame, S., Hagen, T., Cross, D. A., Bax, B., and Reith, A.

D. (2001) FEBS Lett 507(3), 288-94.

59. Cross, D., Alessi, D., Cohen, P., Andjelkovich, M., and Hemmings, B. (1995) Nature

378, 785-789

60. Welsh, G. I., and Proud, C. G. (1993) Biochem. J. 294, 625-629

61. Eldar-Finkelman, H., Argast, G. M., Foord, O., Fischer, E. H., and Krebs, E. G. (1996)

Proc Natl Acad Sci U S A 93(19), 10228-33.

62. Mora, A., Sabio, G., Gonzalez-Polo, R. A., Cuenda, A., Alessi, D. R., Alonso, J. C.,

Fuentes, J. M., Soler, G., and Centeno, F. (2001) J Neurochem 78(1), 199-206.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

31

63. Ding, V. W., Chen, R. H., and McCormick, F. (2000) J Biol Chem 275(42), 32475-81.

64. Hers, I., Tavare, J. M., and Denton, R. M. (1999) FEBS Lett 460(3), 433-6.

65. Smith, D. G., Buffet, M., Fenwick, A. E., Haigh, D., Ife, R. J., Saunders, M., Slingsby, B.

P., Stacey, R., and Ward, R. W. (2001) Bioorg Med Chem Lett 11(5), 635-9.

66. Sutherland, C., Leighton, I. A., and Cohen, P. (1993) Biochem. J. 296, 15-19

67. De Mesquita, D. D., Zhan, Q., Crossley, L., and Badwey, J. A. (2001) FEBS Lett 502(3),

84-8.

68. Williams, R. S., Cheng, L., Mudge, A. W., and Harwood, A. J. (2002) Nature 417(6886),

292-5.

69. Phiel, C. J., Wilson, C. A., Lee, V. M.-Y., and Klein, P. S. (2003) Nature in press

70. Yu, J. Y., Taylor, J., DeRuiter, S. L., Vojtek, A. B., and Turner, D. L. (2003) Mol Ther

7(2), 228-36.

71. Kim, H. S., Skurk, C., Thomas, S. R., Bialik, A., Suhara, T., Kureishi, Y., Birnbaum, M.,

Keaney, J. F., Jr., and Walsh, K. (2002) J Biol Chem 277(44), 41888-96.

72. Fang, X., Yu, S. X., Lu, Y., Bast, R. C., Jr., Woodgett, J. R., and Mills, G. B. (2000) Proc

Natl Acad Sci U S A 97(22), 11960-5.

73. Sutherland, C., and Cohen, P. (1994) Febs Lett 338(1), 37-42

74. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994) J Biol Chem 269(7),

5241-8.

75. Dostmann, W. R. (1995) FEBS Lett 375(3), 231-4.

76. Kuo, C. J., Chung, J., Fiorentino, D. F., Flanagan, W. M., Blenis, J., and Crabtree, G. R.

(1992) Nature 358(6381), 70-3.

77. Villa-Moruzzi, E., Ballou, L. M., and Fischer, E. H. (1984) J Biol Chem 259(9), 5857-63.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

32

78. Jurgensen, S., Shacter, E., Huang, C. Y., Chock, P. B., Yang, S. D., Vandenheede, J. R.,

and Merlevede, W. (1984) J Biol Chem 259(9), 5864-70.

79. Vandenheede, J. R., Yang, S. D., Goris, J., and Merlevede, W. (1980) J Biol Chem

255(24), 11768-74.

80. Park, I. K., and DePaoli-Roach, A. A. (1994) J Biol Chem 269(46), 28919-28.

81. DePaoli-Roach, A. A. (1984) J Biol Chem 259(19), 12144-52.

82. Hemmings, B. A., Resink, T. J., and Cohen, P. (1982) FEBS Lett 150(2), 319-24.

83. Bennecib, M., Gong, C. X., Grundke-Iqbal, I., and Iqbal, K. (2000) FEBS Lett 485(1),

87-93.

84. Tanji, C., Yamamoto, H., Yorioka, N., Kohno, N., Kikuchi, K., and Kikuchi, A. (2002) J

Biol Chem 277(40), 36955-61.

85. Stoothoff, W. H., Bailey, C. D., Mi, K., Lin, S. C., and Johnson, G. V. (2002) J

Neurochem 83, 904-913

86. Dajani, R., Fraser, E., Roe, S. M., Yeo, M., Good, V. M., Thompson, V., Dale, T. C., and

Pearl, L. H. (2003) EMBO J 22, 494-501

87. Eldar-Finkelman, H., and Krebs, E. G. (1997) Proc Natl Acad Sci U S A 94(18), 9660-4.

88. Delcommenne, M., Tan, C., Gray, V., Rue, L., Woodgett, J., and Dedhar, S. (1998) Proc

Natl Acad Sci U S A 95(19), 11211-6.

89. Mora, A., Sabio, G., Risco, A. M., Cuenda, A., Alonso, J. C., Soler, G., and Centeno, F.

(2002) Cell Signal 14(6), 557-62.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

33

Acknowledgements:

We thank Drs. Charles Abrams and Jing Yang for comments on the manuscript and Morrie

Birnbaum for many helpful discussions. We are grateful to James Woodgett for the GSK-3ß-/-

KO cells, Bert Vogelstein and Ken Kinzler and David Kimelman for plasmids, Barry Gumbiner

for purified ß-catenin protein, Peter Davies and Virginia M-Y. Lee for antibodies, and Allen Liu

and Arpine Arzoumanian for technical assistance. This work was supported by a grant to P.S.K.

from the National Institute of Mental Health. P.S.K. is an Assistant Investigator in the Howard

Hughes Medical Institute.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

34

Figure Legends

Figure 1. GID peptides inhibit GSK-3 activity in mammalian cells. A. Neuro2A cells were

co-transfected with pCS2 control (C), GID320-429 (GID-WT), GID(L-P)320-429 , full-length Axin, or

a truncated Axin mutant lacking the RGS domain (∆RGS), together with tau plasmid.

Phosphorylated tau protein was detected by Western blotting (WB) with a phospho-tau specific

antibody (p-tau, top panel) and total tau was detected with T14/46 antibodies (tau, lower panel).

B. Neuro2A cells were transfected with pCS2 control (C), wild-type (WT), and L->P mutants of

full length Axin, GID320-429, and GID380-404. ß-catenin accumulation was detected by WB.

Expression of Axin and GID was detected by WB with myc antibody. HnRNP protein levels are

shown as loading controls. C. Dose-dependent ß-catenin accumulation by GID320-429 and lithium

in Neuro2A cells. ß-catenin was detected by WB as in B.

Figure 2. GID causes ß-catenin accumulation in the cytoplasm and nucleus and activates

the Lef-OT reporter. A. GID induces ß-catenin accumulation in the cytoplasm and nucleus as

visualized by immunofluorescence (IF). NIH3T3 cells were co-transfected with GID380-404,

GID(L-P )380-404, full-length Axin, or pCS2 control (C), together with pEGFP. GFP-expression

(left column) indicates transfected cells. ß-catenin was visualized with rhodamine-conjugated

secondary antibody (red, middle column) and nuclei are stained with DAPI (right column). B,

The number of transfected cells with increased ß-catenin signal were counted in four random

views (each with ~250 cells) and were plotted as a percentage of transfected (GFP positive) cells.

Data are from four independent experiments. Error bars represent standard deviations. C and D,

GID mimics lithium in activating Lef-OT in a dose- and ß-catenin/TCF-dependent manner. 293T


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

35

cells were co-transfected with Lef-OT or Lef-OF (mutated TCF/Lef sites), together with Renilla

luciferase pRL-SV40 (internal control) in the absence or presence of dnTCF. Cells were treated

with LiCl (panel C) or co-transfected with GID380-404 at the plasmid concentrations indicated in D,

or GID(L-P)380-404. Fold activation represents luciferase activity normalized to pCS2 control.

(Data shown are representative of at least four independent experiments with similar results.)

Figure 3. GID peptides directly inhibit GSK-3 activity in vitro. A, Purified recombinant ß-

catenin was added to the GSK-3ß in vitro kinase reactions at 25µg/ml and phosphorylation of ß-

catenin was detected by autoradiography. Recombinant GID320-429-his peptide or LiCl was added at

the indicated concentrations (21 nM is approximately equimolar with GSK-3ß protein in panels A

and B). ß-catenin and GSK-3ß protein levels are visualized by Coomassie staining (middle and

lower panels). In panels A and B, "-S" indicates no substrate, "-E" indicates no GSK-3ß, and "C"

indicates the control reaction in the absence of inhibitors. B, Inhibition of unphosphorylated versus

phosphorylated tau protein: Recombinant tau (non-primed tau) or tau protein that had been pre-

phosphorylated by MAP Kinase (MAPK-primed tau) was added to the GSK-3ß in vitro kinase

reactions at 12.5 µg/ml and phosphorylation of tau was detected by autoradiography. Purified

recombinant GID320-429 -his or LiCl was added at the concentrations indicated. C, PKA activity, as

assayed by [32P]-incorporation into kemptide (50 µM), is inhibited by H-89 (20 µM) but not by

GID380-404 peptide. Similarly p34cdc2/Cdk-1 phosphorylation of Histone-H1 (10 µM) is inhibited by

staurosporine (SST: 1 µM) but not by GID380-404 peptide. (Data shown are representative of at least

four independent experiments.)

Figure 4. Inhibition of GSK-3 activity by GID and GBP/FRAT. A, 293T cells were co-


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

36

transfected with pCS2 control (C), GID380-404, or myc tagged GBP at the concentrations indicated,

together with tau, Lef-OT, and Renilla luciferase (pRL-SV40). The expression levels of GID380-

404 and GBP were detected by WB with myc antibody (bottom panel), tau phosphorylation was

detected by WB with PHF-I antibody (p-tau, top panel) and total tau was detected by WB with

T14/46 antibodies (tau, second panel). Endogenous GSK-3ß protein levels detected by WB are

shown as loading controls. B, Lysates from samples shown in A were assessed for luciferase

activity. Fold Activation represents normalized luciferase activity compared with pCS2 control.

C. In vitro GSK-3ß assay was performed using non-primed tau (12.5 µg/ml) as substrate.

Phosphorylation of tau was detected by autoradiography and quantified as described in

“Experimental Procedures”. Synthetic GID380-404 or FRATide peptides were added at the

concentrations indicated. The IC50 for GID380-404 peptide is approximately 50nM and IC50 for

FRATide is approximately 160nM. (Data shown are representative of at least three independent

experiments with similar results.)

Figure 5. GID induces serine-9 phosphorylation of GSK-3ß mimicking lithium. A-C, serine-

9 phosphorylation of GSK-3ß (p-GSK-3ß (S9)) was detected by WB with phospho-GSK-3ß

(serine-9) antibody. GSK-3ß protein levels are shown as loading controls. A, Lithium treatment

causes rapid and robust phosphorylation of GSK-3ßser9. 293T cells were treated with LiCl

(20mM) for the times indicated. B, GID but not FL Axin causes GSK-3ßser9 phosphorylation,

mimicking lithium. 293T cells were transfected with pCS2 control (C), wild-type(WT) and L390-

>P mutants of GID320-429, GID380-404 and full length Axin, or were treated with lithium as in A for 4

hours (as positive control). Endogenous ß-catenin levels (third panel) and expression of myc-

tagged, transfected proteins (fourth panel) were detected by WB. C. 293T cells were treated with


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

37

LiCl or transfected with GID380-404 or pCS2 control (C) at the concentrations indicated. Cells were

harvested after 4 hours of LiCl treatment or 24 hours after transfection. Expression of GID380-404

was detected by WB with myc antibody (third panel). (Data shown are representative of at least

four independent experiments done in duplicate with similar results.)

Figure 6. Small molecule GSK-3 inhibitors cause increased serine-21/9 phosphorylation. A.

Neuro2A cells were treated with LiCl (Li), Rottelerin (Rott), Quercetin (Quer), Flavopiridol

(Flavo), Ro31-8220 (Ro31), or DMSO (vehicle control, C) for 2 hours at concentrations

indicated. GSK-3ß serine-9 phosphorylation (p-GSK-3ß(S9)) was detected by WB with the

phospho-serine-9-GSK-3ß antibody. GSK-3ß protein levels are shown as loading control. B,

Time course: Neuro2A cells were treated with Rottlerin (1µM), LiCl (20mM) or Ro31-8220

(50nM) for the times indicated. Phosphorylation of GSK-3αser21 (p-GSK-3α(S21)) and GSK-3ß

ser9 (p-GSK-3ß(S9)) was detected by WB with an antibody that recognizes both GSK-3α and ß

phosphorylated at their respective N-termini (upper panel) . GSK-3α/ß protein levels were

detected by WB with an antibody that recognizes both isoforms. C, Neuro2A cells were treated

with TDZD, LiCl, valproic acid (VPA), or DMSO (vehicle control, C) for 2 hours at the

concentrations indicated and WB performed as in A. D, Neuro2A cells were treated with TDZD

(50µM) or LiCl (20mM) for the times indicated and WB performed as in B.

Figure 7. Loss of GSK-3 expression leads to increased N-terminal phosphorylation of GSK-

3. A, CHO cells were transfected with siRNAs targeted against pGL3-luciferase (GL3; control

siRNA), GSK-3α or ß. GSK-3 protein levels and N-terminal phosphorylation were detected by

WB. "Un" represents untransfected cells. HnRNP protein levels are shown as loading controls.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

38

B, Mouse embryonic fibroblasts (MEF) from wild-type (GSK-3ß+/+ , lane 1-3) or GSK-3ß knock-

out mice (GSK-3ß-/-, lane 4-6) were treated with 20mM of LiCl for the times indicated. WB for

GSK-3α and ß and for phospho-GSK-3α and ß were performed as in Fig 6B. HnRNP protein

levels are shown as loading controls. (Data shown are representative of at least four independent

experiments with similar results.)

Figure 8. Increased phosphorylation of GSK-3 after lithium treatment is not mediated

through activation of Akt, p90rsk2, PKA, or p70S6K. A. Diagram showing phosphorylation of

GSK-3α/ß by kinase(s), such as Akt, p90rsk2, PKA, or p70S6K that could, in principle, be

antagonized by GSK-3 and therefore activated by GSK-3 inhibitors such as lithium. B, Neuro2A

cells were treated with LiCl (20mM) or insulin (Ins., 1µg/ml) for the times indicated with or

without LY294002 (LY, 50µM) pretreatment. Phosphorylation of Akt at serine-473 (which

reflects activation) or GSK-3ß at serine-9 was detected by WB with the respective phospho-

specific antibodies. Endogenous GSK-3ß levels (third panel) are shown as loading controls. C,

NIH3T3 cells were treated with LiCl (20mM) or PDGF (50µg/ml) for the times indicated with or

without LY pretreatment (LY: lanes 4, 7, 9, and 11). Phosphorylation of Akt was detected as in

B. Serine-21/9 phosphorylation of GSK-3α/ß was detected by WB with the phospho-GSK-3α/ß

(serine-21/9) antibody. D, 293 T cells were treated with lithium and blotted for phospho-Akt and

phospho-GSK-3ß as in B. E, Time-course for phosphorylation of p90rsk2 versus GSK-3ß

following lithium treatment: Neuro2A cells were treated with LiCl (20mM) or Rottlerin (1µM)

for the times indicated. Phosphorylation of p90rsk2 (indicative of activation) was detected by WB

with phospho-p90rsk (ser381) antibody. F, Li-induced GSK-3 phosphorylation in the presence of

PKA and p90rsk2 inhibitors: Neuro2A cells were pretreated with the PKA inhibitor Rp-cAMPS


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

39

(Rp, 20µM) and BIS-III (BIS; 1µM), which inhibit the p90rsk2, for 30min, followed by LiCl

(20mM) treatment for 2 hours. (Data shown are representatives of at least four independent

experiments).

Figure 9. GSK-3 autoregulation of serine-9 phosphorylation via the GSK-3 activated PP-

1/I-2 complex. A, Model showing GSK-3 activation of protein phosphatase-1 (PP-1) through

phosphorylation and inhibition of the inhibitory subunit I-2. If PP-1 in turn dephosphorylates

GSK-3, then lithium should cause increased GSK-3 N-terminal phosphorylation by inhibiting

GSK-3-mediated activation of PP-1. B, PP-1α(1.25U) was incubated with serine-9-

phophorylated GSK-3ß with or without I-2 (1µM). ATP was added at 0.2mM to activate GSK-

3ß. LiCl (20mM) or GID320-429-his peptide (105 nM) was added as indicated. GSK-3ß and

phospho-GSK-3ß were detected by WB as in previous figures. C. LiCl (20mM) or GID320-429-his

(105 nM) was added in the absence of I-2 to verify that these inhibitors do not inhibit PP-1

directly. PP-1 protein levels are visualized by Coomassie staining. D. 293T cells were

transfected with pCS2 control (C) or myc-tagged mouse I-2 and were harvested after 24 hours.

GSK-3ß and phospho-GSK-3ß(S9) were detected as above and I-2 expression was detected with

myc antibody. (Data shown are representative of at least four independent experiments with

similar results.)


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

by guest on August 27, 2020http://www.jbc.org/Downloaded from

http://www.jbc.org/


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

by guest on August 27, 2020http://www.jbc.org/Downloaded from

http://www.jbc.org/


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Fang Zhang, Christopher J. Phiel, Laura Spece, Nadia Gurvich and Peter S. Kleinlithium: Evidence for autoregulation of GSK-3

Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to

published online June 7, 2003J. Biol. Chem.

10.1074/jbc.M212635200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M212635200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M212635200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/06/07/jbc.M212635200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2003/06/07/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2003/06/07/jbc.M212635200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

Inhibitory phosphorylation of glycogen synthase kinase-3 ... · 6/7/2003 · Lithium salts have...

Documents

Transcript of Inhibitory phosphorylation of glycogen synthase kinase-3 ... · 6/7/2003 · Lithium salts have...