Báo cáo khoa học: A novel glycogen-targeting subunit of protein phosphatase 1 that is regulated by insulin and shows differential tissue distribution in humans and rodents pdf

A novel glycogen-targeting subunit of protein phosphatase 1
that is regulated by insulin and shows differential tissue
distribution in humans and rodents
Shonagh Munro1, Hugo Ceulemans2, Mathieu Bollen2, Julie Diplexcito1 and Patricia T.W. Cohen1
1 Medical Research Council Protein Phosphorylation Unit, University of Dundee, UK
2 Katholieke Universiteit Leuven, Faculteit Geneeskunde, Afdeling Biochemie, Belgium

Keywords
diabetes; glycogen metabolism; glycogen
synthase; insulin; PP1
Correspondence
P. T. W. Cohen, MRC Protein
Phosphorylation Unit, School of Life
Sciences, MSI ⁄ WTB Complex, University of
Dundee, Dow Street, Dundee DD1 5EH, UK
Fax: +44 1382 223778
Tel: +44 1382 344240
E-mail:
(Received 23 September 2004, revised 16
December 2004, accepted 26 January 2005)
doi:10.1111/j.1742-4658.2005.04585.x

Stimulation of glycogen-targeted protein phosphatase 1 (PP1) activity by
insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis. The glycogen-targeting subunits of PP1, GL and R5 ⁄ PTG, are downregulated in
the livers of diabetic rodents and restored by insulin treatment. We show
here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver. The phosphatase activity associated with R3E is slightly higher than that associated with
R5 ⁄ PTG and it is downregulated in streptozotocin-induced diabetes by 60–
70% and restored by insulin treatment. Surprisingly, although mRNA for
R3E is most highly expressed in rat liver and heart muscle, with only low
levels in skeletal muscle, R3E mRNA is most abundant in human skeletal
muscle and heart tissues with barely detectable levels in human liver. This

species-specific difference in R3E mRNA expression has similarities to the
high level of expression of GL mRNA in human but not rodent skeletal
muscle. The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents
and humans.

Insulin-stimulated glycogen synthesis is decreased in
type 2 diabetes [1,2]. One of the routes by which insulin stimulates this pathway is through activation of the
rate-limiting enzyme, glycogen synthase (GS), via the
phosphatidylinositol-3-kinase ⁄ protein kinase B pathway, which leads to the inhibition of glycogen synthase
kinase 3 (GSK3) [3,4]. Activation of GS results from a
net dephosphorylation of serine residues that are phosphorylated by GSK3 and dephosphorylated by glycogen-associated protein phosphatase 1 (PP1) [5–8]. In
order to determine how insulin modifies GS activity, it
is therefore crucial to understand the mechanisms by
which insulin may activate glycogen-targeted PP1. The

latter mainly exists as heterodimeric complexes of the
catalytic subunit, PP1c, bound to a regulatory subunit
[9]. In striated muscles the most abundant glycogenbinding subunit GM (124–126 kDa, encoded by the
gene PPP1R3A) targets PP1c to the sarcoplasmic reticulum as well as to glycogen particles [10–12]. A much
smaller protein, GL (33 kDa, encoded by the gene
PPP1R3B), is the most abundant glycogen-targeting
subunit of PP1 in liver, although it is only 23% identical to the N-terminal region of GM [13,14]. Two other
glycogen-binding subunits, R5 ⁄ PTG (36 kDa, encoded
by PPP1R3C) with  40% identity to GL and R6
(33 kDa, encoded by PPP1R3D) with  30% identity

Abbreviations
GL, hepatic glycogen-targeting subunit of PP1 encoded by the gene PPP1R4(3B); GM (also termed RGL), skeletal muscle glycogen-targeting
subunit of PP1 encoded by the gene PPP1R3(3A); GS, glycogen synthase; GSK3, glycogen synthase kinase-3; GSP, glycogen synthase
phosphatase; GST, glutathione S-transferase; MBP, maltose-binding protein; NCBI, National Center for Biotechnology Information USA;

PCR, PP1, protein phosphatase 1; PP1c, protein phosphatase 1 catalytic subunit; R5 (also termed PTG), regulatory subunit of PP1 encoded
by the gene PPP1R5(3C); R6, regulatory subunit of PP1 encoded by the gene PPP1R6(3D).

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FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS


S. Munro et al.

to GL have a wide tissue distribution [15–17]. Interestingly, although GL is expressed at only very low levels
in rodent skeletal muscles, it is found in human skeletal muscles at levels comparable with those in human
liver [18]. The four glycogen-targeting subunits bind
to PP1c via a short highly conserved motif (-RVXF-).
This motif is also responsible for the interaction of
many other regulatory subunits with PP1, explaining
why the binding of targeting regulatory subunits to
PP1c is mutually exclusive [19]. However, certain
inhibitor proteins have also been noted to form ternary
structures with PP1c-targeting subunit complexes
[10,20–22]. In addition to the PP1 and glycogen-binding motifs, the PP1 glycogen-regulatory subunits possess a motif for the interaction with substrates [23].
The glycogen-targeting subunits can modulate the
activity of PP1c towards different substrates; for example, GL enhances PP1c activity towards GS while suppressing its activity towards phosphorylase. There is
evidence that PP1-GM and PP1-GL may be regulated
acutely by insulin. Assay of PP1 following insulin infusion of skeletal muscle and immunopelleting of
PP1-GM showed a 1.5–2-fold increase in phosphatase
activity with insulin [24]. In GM null mice, this activity
was absent and GS could not be fully activated by
insulin [24]. In contrast, studies on an independently
derived GM null mouse model found that insulin activation of GS was in the normal range, indicating that

the PP1-GM is not required for the insulin activation
of GS [25]. These workers postulated the existence of a
novel insulin-activated form of glycogen-targeted PP1
[25]. In the case of hepatic glycogen-targeted PP1,
insulin is thought to exert its acute activating effect on
PP1-GL mainly through modulation of cAMP levels
and decrease of phosphorylase a, which is a potent
inhibitor of hepatic glycogen synthase phosphatase
(GSP) activity [26–32]. Phosphorylase a binds to 16
amino acids at the extreme C-terminus of GL, a sequence that is absent from the other three glycogentargeting subunits [18,31]. R5 ⁄ PTG and R6 ⁄ PPP1R3D
are not known to be acutely regulated by insulin. Insulin exerts a longer term regulation on hepatic GL and
R5 ⁄ PTG [33,34]. Diabetic rats exhibit a loss of hepatic
glycogen-bound synthase phosphatase activity that can
be restored by insulin administration [35,36]. The main
underlying defects are decreased expression of the two
PP1 glycogen-targeting subunits, GL and R5 ⁄ PTG, in
the diabetic state [33,34]. Downregulation of both the
protein and mRNA levels of hepatic GL and R5 ⁄ PTG
are restored by insulin treatment, but the skeletal muscle R5 ⁄ PTG level is not altered by insulin [33,34,37].
The expression of hepatic R6 ⁄ PPP1R3D is also unaffected in diabetic animals [34].
FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS

Novel glycogen-targeting subunit of PP1

Ceulemans et al. [38], undertook a bioinformatic
approach in order to trace the evolution of regulatory
subunits of PP1. Searching completed genome
sequences, with the sequences of known PP1 regulatory subunits, including the conserved PP1 and glycogen-binding regions of the glycogen-targeting subunits,
they identified nine new potential regulatory subunits
of PP1. Of these nine sequences, three were deduced

to encode for putative human glycogen-targeting subunits, and were given the nomenclature PPP1R3E,
PPP1R3F and PPP1R3G. These potential human proteins all contained the canonical -RVXF- motif that
mediates interaction with PP1, as well as putative modules for targeting to glycogen and facilitating interaction with PP1 substrates such as GS. Here we show
that the phosphatase activity of one of these novel targeting subunits, R3E, is under long-term control by
insulin in rodent liver while being virtually absent from
rodent skeletal muscle; yet surprisingly, PPP1R3E
mRNA is found at appreciable levels in human skeletal
muscle.

Results
Cloning of human PPP1R3E and PPP1R3G
from human cDNA libraries
Interrogation of the NCBI databases revealed no fulllength mammalian cDNAs sharing similarities to the
human genomic sequences PPP1R3E, PPP1R3F and
PPP1R3G (Accession nos: AL049829, NM_033215,
AL035653) with chromosomes locations 14q11.2,
Xp11.23 and 6p24.3-25.3, respectively. In order to
establish whether these sequences were functional
genes or pseudogenes, attempts were made to amplify
their putative cDNAs from human libraries, using
primers designed from the genomic sequences. For
PPP1R3E, a single cDNA of  800 bp was amplified
by two rounds of polymerase chain reaction (PCR)
from both human testis and brain cDNA libraries. The
size of these products was consistent with the expected
size for a putative sequence for a glycogen-targeting
subunit of PP1 assuming the genomic sequence has
two coding exons separated by one intron (Fig. 1A). A
cDNA corresponding to the genomic sequence of
PPP1R3G was obtained by PCR from the human

brain library, but no full-length cDNA products were
obtained for PPP1R3F. Subcloning and sequencing of
the PCR products confirmed the identity of the testis
and brain cDNAs for PPP1R3E and showed that the
protein translated from this sequence was composed of
279 amino acids with a predicted molecular mass of
30.6 kDa (Fig. 1B). The PPP1R3G cDNA sequence
1479


Novel glycogen-targeting subunit of PP1

A

S. Munro et al.

Gene
-272

417

719

E1

1279 1713 1819

E2

3036 3176

E3

3660

E4

6656

E5

mRNA
1 (ATG)

E1

837 (STOP)

E2

E3 E4

E5

B
-105 gaagcggacccagcgacttctgcgctgacgcggggcgggcgggagagaggaagagaggggagcgcggtggcgctgcgagctggccccgccggggaaggggctgcc -1
1
1

ATG TCC CGT GAG CGG CCC CCG GGC ACC GAC ATT CCC CGC AAC CTG AGC TTC ATC GCC GCG CTA ACG GAG CGC GCC

M
S
R
E
R
P
P
G
T
D
I
P
R
N
L
S
F
I
A
A
L
T
E
R
A

75
25

76

26

TAC TAC CGT AGC CAG CGG CCC AGC CTC GAG GAG GAG CCG GAG GAG GAG CCA GGC GAG GGC GGG ACG CGG TTC GGG
Y
Y
R
S
Q
R
P
S
L
E
E
E
P
E
E
E
P
G
E
G
G
T
R
F
G

150

50

151
51

GCC CGA TCC CGC GCT CAC GCA CCG AGT CGG GGC CGC CGG GCC CGA TCT GCA CCA GCC GGA GGC GGC GGG GCC CGG
A
R
S
R
A
H
A
P
S
R
G
R
R
A
R
S
A
P
A
G
G
G
G
A

R

225
75

226
76

GCG CCC CGC AGC CGT AGC CCA GAC ACC CGC AAG AGA GTG CGT TTC GCC GAC GCA CTG GGG TTG GAG CTG GCT GTC
A
P
R
S
R
S
P
D
T
R
K
R
V
R
F
A
D
A
L
G
L

E
L
A
V

300
100

301
101

GTG CGC CGC TTC CGT CCC GGT GAG CTG CCC CGG GTG CCC CGC CAC GTG CAG ATC CAA TTG CAG AGG GAC GCC CTC
V
R
R
F
R
P
G
E
L
P
R
V
P
R
H
V
Q
I

Q
L
Q
R
D
A
L

375
125

376
126

CGC CAC TTC GCG CCC TGC CAG CCC CGC GCC CGC GGC CTC CAG GAG GCG CGC GCC GCC CTG GAG CCG GCC AGC GAG
R
H
F
A
P
C
Q
P
R
A
R
G
L
Q
E

A
R
A
A
L
E
P
A
S
E

450
150

451
151

CCC GGC TTC GCC GCC CGC TTG CTG ACG CAG CGC ATC TGC CTG GAA CGC GCC GAG GCG GGC CCG CTG GGC GTG GCC
P
G
F
A
A
R
L
L
T
Q
R
I

C
L
E
R
A
E
A
G
P
L
G
V
A

525
175

526
176

GGG AGC GCG CGC GTG GTG GAC CTG GCC TAC GAG AAG CGC GTG AGC GTG CGC TGG AGC GCC GAC GGC TGG CGG AGC
G
S
A
R
V
V
D
L
A

Y
E
K
R
V
S
V
R
W
S
A
D
G
W
R
S

600
200

601
201

CAA CGC GAG GCG CCA GCC GCC TAC GCC GGT CCG GCC CCG CCC CCG CCG CGC GCC GAC CGC TTC GCC TTC CGC CTG
Q
R
E
A
P
A

A
Y
A
G
P
A
P
P
P
P
R
A
D
R
F
A
F
R
L

675
225

676
226

CCC GCG CCG CCG ATT GGG GGC GCC CTG CTC TTC GCC TTG CGC TAC CGT GTG ACA GGT CAC GAG TTC TGG GAC AAC
P
A
P

P
I
G
G
A
L
L
F
A
L
R
Y
R
V
T
G
H
E
F
W
D
N

750
250

751
251

AAC GGC GGC CGT GAC TAT GCT CTA CGT GGG CCC GAG CAC CCG GGC AGT GGC GGA GCT CCG GAG CCG CAG GGC TGG

N
G
G
R
D
Y
A
L
R
G
P
E
H
P
G
S
G
G
A
P
E
P
Q
G
W

825
275

826

276

ATC CAC TTT ATC TGA gacgaggcgcctgcggccgacggcggaaaacaccaaaggcacccgggggcggggcgacccgatgtggcggggaggagtag 920
I
H
F
I
*
279

921

gagagaccaggattggcgggagcggtccaagggagtc

957

Fig. 1. (A) Diagram of human PPP1R3E mRNA compared with PPP1R3E gene (Accession no: ENSG00000129525). Nucleotide numbers at
the start and end of each exon are given relative to the first nucleotide of the initiating methionine codon. The exon ⁄ intron structure within
the coding region was determined experimentally by PCR of cDNA libraries, whereas that for the untranslated region is predicted from the
genomic sequence and partial cDNAs in the database. (B) Human PPP1R3E cDNA and the encoded protein determined by PCR of human
brain and testis cDNA libraries. The PP1 binding motif is underlined, glycogen-targeting domain is double underlined, and the substrate-binding sequence is underscored by a wavy line. Oligonucleotides primers used for PCR are indicated by arrows.

was verified as being identical to the genomic sequence
with one coding exon specifying a protein of 358
amino acids and a molecular mass of 38 kDa (data
not shown).
Comparison of the glycogen-targeting subunits
of PP1
Searching mouse and rat genomic sequences in the
NCBI databases identified predicted rodent cDNAs

from genes homologous to human PPP1R3E and
1480

PPP1R3G. The encoded rat and mouse R3E proteins
share around 97% amino acid identity and are 89%
identical to their human orthologue, indicating that
this regulatory protein is very well conserved in mammals (Fig. 2A). R3G is slightly less highly conserved;
the rodent orthologues are 90% identical and they are
11 amino acids shorter than their human orthologue,
sharing 67% identity (Fig. 2B). A phylogenetic tree
depicting the relationship between known glycogen-targeting subunits of PP1 and the novel glycogen-targeting subunits is shown in Fig. 2D. Although all seven
FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS


S. Munro et al.

Novel glycogen-targeting subunit of PP1

A

C

MOUSE R3E
RAT
R3E
HUMAN R3E

1 MSPERPPRTDIPRNLSFIAALTERAYYRSQRPSLEEESEEEPGEGGTRPGARSRAHVPG
1 MSHERPPRNDIPRNLSFIAALTERAYYRSQRPSLEEESEEEPGEGGTRPGARSRAHVPG
1 MSRERPPGTDIPRNLSFIAALTERAYYRSQRPSLEEEPEEEPGEGGTRFGARSRAHAPS

MOUSE R3E
RAT
R3E
HUMAN R3E

60 RGRRARSAPAGGGGARTARSRSPDTRKRVRFADALGLELAVVRRFRPGEPPRVPRHVQV
60 RGRRARSAPAGGGGARTARSRSPDTRKRVRFPDALGLELAVVRRFRPGEPPRVPRHVQV
60 RGRRARSAPAGGGGARAPRSRSPDTRKRVRFADALGLELAVVRRFRPGELPRVPRHVQI

MOUSE R3E 119 QLQRDALRHFAPCPPRARGLQEARVALEPALEPGFAARLQAQRICLERADAGPLGVAGS
RAT
R3E 119 QLQRDALRHFAPCPPRTRGLQDARIALEPALEPGFAARLQAQRICLERADAGPLGVAGS
HUMAN R3E 119 QLQRDALRHFAPCQPRARGLQEARAALEPASEPGFAARLLTQRICLERAEAGPLGVAGS

PP1 binding motif
GM/ RGL/R3A
GL/R3B
R5/PTG/R3C
R6/R3D
R3E
R3F
R3G

60
58
81
99
84
167

128

GTRRVSFAD
VKKRVSFAD
AKKRVVFAD
QKLRVRFAD
TRKRVRFAD
APRRVLFAD
CKKRVQFAD

Glycogen binding domain

MOUSE R3E 178 ARVLDLAYEKRVSVRWSADGWRSLRESPASYAGPAPSPPRADRFAFRLPAPPVGGTLLF
RAT
R3E 178 ARVLDLAYEKRVSVRWSADGWRSLRESPASYAGPAPAPPRADRFAFRLPAPPVGGALLF
HUMAN R3E 178 ARVVDLAYEKRVSVRWSADGWRSQREAPAAYAGPAPPPPRADRFAFRLPAPPIGGALLF

GM/ RGL/R3A
GL/R3B
R5/PTG/R3C
R6/R3D
R3E
R3F
R3G

MOUSE R3E 237 ALRYRVTGREFWDNNGGRDYALLGPEHPAGAGAAEPQGWIHFI 279
RAT
R3E 237 ALRYRVTGREFWDNNGGRDYALLGPEHPGGAGAAEPQGWIHFI 279
HUMAN R3E 237 ALRYRVTGHEFWDNNGGRDYALRGPEHPGSGGAPEPQGWIHFI 279

Substrate binding domain

B
Rat
R3G
Mouse R3G
Human R3G

1 MEASGEQLHRSEASSSTSSEDPPPAEELSVPEVLCVESG-----TSEVPI
1 MDPSGEQLHRSEASSSTSSGDPQSAEELSVPEVLCVESG-----TSETPI
1 MEPIGARLS-LEAPGPAPFREAPPAEELPAPVVPCVQGGGDGGGASETPS

Rat
R3G
Mouse R3G
Human R3G

46 PDDQLQDRLLSAQKVAALPEQEELQEYRR-SRVRSFSLPADPILQAAKLL
46 PDAQLQDRPLSPQKGAALPEQEELQEYRR-SRARSFSLPADPILQAAKLL
50 PDAQLGDRPLSPKEEAAPQEQEELLECRRRCRARSFSLPADPILQAAKFL

GM/ RGL/R3A
GL/R3B
R5/PTG/R3C
R6/R3D
R3E
R3F
R3G

144

146
171
191
176
300
235

219
221
246
267
248
407
339

GIIRVLNVSFEKLVYVRMSLDDW
GTVKVQNLAFEKTVKIRMTFDTW
GTVKVKNVSFEKKVQIRITFDSW
GTVRVCNVAFEKQVAVRYTFSGW
GSARVVDLAYEKRVSVRWSADGW
GLVRVLNRSFEKAVHVRASHDGW
GSGRVLSCPGPRAVTVRYTFTEW

WSNNNGTNY
WDSNRGKNY
WDNNDGQNY
WDNNDHRDY
WDNNGGRDY
WANNHGRNY
WDNNAGANY

D
hR3F mR3F
GM/RGL

Rat
R3G 95 QQRQQ-----AGQPSSEGGEPAGDCCSKCKKRVQFADSLGLSLASVKHFS
Mouse R3G 95 QQRQQ-----AGQPSSEGGAPAGDCCSKCKKRVQFADSLGLSLASVKHFS
Human R3G 100 QQQQQQAVALGGEGAEDAQLGPGGCCAKCKKRVQFADTLGLSLASVKHFS

Rat
R3G 140 EAEEPQVPPAVLSRLHSFPLRAEDLQQLGELLAVAKVPAPLLTPRAQLRP
Mouse R3G 140 EAEEPQVPPAVLSRLHSFPLRAEDLQQLGGLLAVATMPDPLLVPCARLRP
Human R3G 150 EAEEPQVPPAVLSRLRSFPMRAEDLEQLGGLLAAAAVAAPLSAPPSRLRP

Rat
R3G 190 LFQLPGLIAAEERLRRQRVCLERVQCSQPPRAEVTGSGRVISCPGPRAVA
Mouse R3G 190 HFQLPELRAAEERLRRQRVCLERVQCSQPPRAEVTGSGRVISCPGPRAVA
Human R3G 200 LFQLPGPSAAAERLQRQRVCLERVQCSTASGAEVKGSGRVLSCPGPRAVT

rbR3A mR3A
hR3A

R6
mR3DrR3D
hR3D

mR3E
rR3E
hR3E

hR3C
R5/PTG rR3C
mR3C
hR3B
rR3B mR3B

mR3G
hR3G rR3G

GL

Rat
R3G 240 VRYTFTEWRTFLDVPAELHPESLEPLSP-VRSGNSGPGAEDSEGEPGTER
Mouse R3G 240 VRYTFTEWRTFLDVPAELDPESLEPLPP-LQSGDSGSKAEDSEEGPGTER
Human R3G 250 VRYTFTEWRSFLDVPAELQPEPLEPQQPEAPSGASEPGSGDAKKEPGAEC

Rat
R3G 289 FCFSLCLPPGLQPKEGEDADTWGVAIHFAVCYRCEQGEYWDNNEGANYTL
Mouse R3G 289 FHFSLCLPPGLQPKEGEDAGAWGVAIHFAVCYRCEQGEYWDNNEGANYTL
Human R3G 300 FHFSLCLPPGLQPEDEEDADERGVAVHFAVCYRCAQGEYWDNNAGANYTL

Rat
R3G 339 RYVCSTDPL 347
Mouse R3G 339 RYVCSTDPL 347
Human R3G 350 RYARPADAL 358

Fig. 2. Amino acid alignment of human proteins with their rat and mouse homologues (A) R3E, (B) R3G. Identities are shaded in black and
similarities are shaded in grey. The PP1-binding motif is indicated by a single underline. The sequences were aligned using CLUSTALW (http://
www.clustalw.genome.ad.jp/) and shading was performed using BOXSHADE (v3.21 K.Hofmann and M.Baron). NCBI Accession nos for predicted cDNAs are: XM_193763 (mouse R3E), XM_344406 (rat R3E), XM_225280 (rat R3G). Mouse R3G cDNA Accession no. is AK049829. (C)

Amino acid alignment of the conserved regions of the glycogen-targeting subunits of PP1. Identification of the PP1-binding motif was described in Egloff et al. [43], the glycogen-binding domain in [15,31,44] and the substrate binding domain in [23,45]. (D) Phylogenetic relationship between the glycogen-targeting subunits of PP1. The unrooted tree is derived by the neighbour-joining method in CLUSTAL W from
pairwise sequence distances between the conserved PP1, glycogen and substrate-binding domains (corresponding to amino acids 85–258 of
R3E) of human (h), mouse (m), rat (r) and rabbit (rb) glycogen-targeting subunits. The proteins aligned and their database Accession nos are
R3A(PPP1R3AG ⁄ RGL) NP_002702 (h), NP_536712 (m), A40801 (rb); R3B(PPP1R3B ⁄ GL) [18] and NP_078883 (h), NP_808409 (m), NP_620267
(r); R3C(PPP1R3C ⁄ R5 ⁄ PTG) NP_005389 (h), NP_058550 (m), XP_220048 (r); R3D(PPP1R3D ⁄ R6) Y18206 ⁄ NP_006233 (h), XP_141580 (m),
XP_230940 (r); R3E(PPP1R3E) this study (h, m, r); R3F(PPP1R3F) XP_372210 (h), AAH59275 (m); R3G(PPP1R3G) this study (h, m, r).

FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS

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Novel glycogen-targeting subunit of PP1

Pancreas

Kidney

Skeletal muscle

Liver

Placenta

Lung

Brain

Human PPP1R3E mRNA blot

Heart

A

S. Munro et al.

9.5 kb
7.5 kb

7.2 kb
5.9 kb

4.4 kb

Tissue distribution of PPP1R3E and PPP1R3G
mRNA

2.4 kb
1.35 kb

β-actin
α-actin

2.0 kb
1.8 kb

Kidney
Testis

Skeletal muscle

Lung
Liver

Spleen

Brain

Rat PPP1R3E mRNA blot

Heart

B

9.5 kb
7.5 kb

6.0 kb
5.0 kb
4.5 kb

4.4 kb
2.4 kb
1.35 kb
2.0 kb
1.8 kb

β-actin
α-actin

9.5 kb
7.5 kb

Pancreas

Kidney

Skeletal muscle

Liver

Placenta

Lung

Brain

Human PPP1R3G mRNA blot

Heart

C

2.4 kb
1.35 kb

0.24 kb

1482

The human PPP1R3E cDNA probe hybridized to two
mRNA species on a human multiple tissue northern
blot with sizes 7.2 and 5.9 kb (Fig. 3A). These transcripts were predominantly present in skeletal muscle
and heart, although the smaller transcript was also
present in pancreas and placenta and was detectable at
very low levels in liver and kidney. The sizes and the
tissue distribution of these transcripts are not consistent with those encoding any of the other characterized
glycogen-targeting subunits. In addition, the extremely
low level of sequence similarity at the nucleotide level
implies that cross-hybridization with the mRNAs for
the other subunits is unlikely. Hybridization of a northern blot with the human PPP1R3G cDNA probe
revealed a single PPP1R3G mRNA transcript of
 9 kb that was present exclusively in brain (Fig. 3C).
Unfortunately, attempts to amplify mouse or rat
PPP1R3E cDNA from tissue specific libraries were
unsuccessful. However, the rat PPP1R3E exons showed
a high level of conservation (86% identity) with the
coding region of human PPP1R3E cDNA. This coupled with the lack of sequence similarity to the coding
regions of other glycogen-targeting subunits, allowed
the human cDNA probe to be used to establish the tissue distribution of rat PPP1R3E mRNA on a northern
blot (Fig. 3B). Following a series of stringent washes
and autoradiography, the probe hybridized predominantly to 6.0 kb and 5.0 kb mRNA species in heart

~ 9.0 kb

4.4 kb

2.0 kb
1.8 kb

human subunits and their rodent orthologues possess
known or putative PP1, glycogen and substrate-binding domains (Fig. 2C), no two subunits share more
than 40% amino acid identity. Despite this, each glycogen-targeting subunit is particularly well conserved
between rodents and humans, suggesting that each
subunit may serve an important, nonredundant function in mammals.

β-actin
α-actin

Fig. 3. Tissue distribution of (A) human PPP1R3E mRNA (B) rat
PPP1R3E mRNA (C) human PPP1R3G mRNA. Blots contained
 2 lg of poly(A)+ RNA from different tissues. The upper panels of
(A) and (B) were hybridized with a probe corresponding to the
entire coding region (837 bp) of human PPP1R3E and the upper
panel of (C) was hybridized with a probe corresponding to the
entire coding region (1074 bp) of human PPP1R3G. Following autoradiography, the membranes were stripped in 0.5% (w ⁄ v) SDS at
100 °C for 5 min and subsequently re-probed with a b-actin in order
to assess whether equal amounts of the samples were loaded. In
heart and skeletal muscle the b-actin probe cross-hybridizes with
a-actin.

FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS


S. Munro et al.

Novel glycogen-targeting subunit of PP1

A

B

Fig. 4. Specificity and characterization of
PPP1R3E antibodies. (A) Recognition of
0.1–10 ng of bacterially expressed GST-R3E
by anti-R3E(8–23) and anti-GST-R3E(1–98)
sera. Both antibodies were used at a
concentration of 0.2 lgỈmL)1. (B) Specificity
of anti-R3E(1–98) sera for R3E. The
immunoblot of several glycogen-targeting
subunits was probed with 0.2 lgỈmL)1
affinity purified antibody. Lane 1, rat liver
glycogen pellet; lane 2, rat liver lysate; lane
3, 1 ng GST-R3E(full-length); lane 4, 2 ng
GST-R3E, lane 5, 5 ng GST-R3E; lane 6,
100 ng GST-GM(1–243); lane 7, 100 ng GSTGL; lane 8, 100 ng GST-R5 ⁄ PTG; lane 9,
100 ng GST-R6. In the lower panel, the blot
was stripped and reprobed with ant-GST
sera to show the loading of the samples.

C

and to a 4.5 kb RNA mRNA in liver. Surprisingly, the
probe hybridized only weakly to the 6.0 and 5.0 kb
transcripts in skeletal muscle. The 5.0 kb transcript was
also present in brain, spleen, lung, liver, kidney and
testis, albeit at very low levels.
R3E protein is present in the rat liver glycogen
fraction and phosphatase activity associated with
R3E is higher than that associated with R5/PTG

Anti-R3E(8–23) sera were raised against amino acids
8–23 in the N-terminus of human R3E, as this is the
region that shares no similarity with other glycogentargeting subunits. These antibodies and anti-GSTR3E(1–98) sera recognized as little as 0.2 ng of bacterially expressed GST-R3E(full length, human) (Fig. 4A).
Anti-GST-R3E(1–98) was virtually specific for R3E as
it did not recognize 100 ng of GM, GL, R6 or
R5 ⁄ PTG (Fig. 4B). The peptide antibody was extremely specific as it did not cross-react with 100 ng of
GM, GL, R5 ⁄ PTG or R6 (data not shown).
FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS

The presence of a fairly well-conserved glycogenbinding motif in R3E suggested that it may interact
with glycogen. To test this hypothesis, a rat liver
lysate, microsomal fraction and a glycogen fraction
were prepared and the proteins in these fractions
separated by SDS ⁄ PAGE, transferred to nitrocellulose
and immunoblotted. A single R3E band was detected
in the glycogen fraction, which was consistent with the
predicted size of R3E ( 31 kDa) (Fig. 5A). This band
was sometimes detectable at low levels in rat liver
lysates (Figs 4B and 5A).
In order to establish whether R3E could bind (and
therefore be regulated allosterically by phosphorylase a, 4 lg of GST-R3E was transferred to nitrocellulose membrane and tested for its ability to bind to
[32P]phosphorylase a [31]. The 32P-labelled phosphorylase a was found to bind to GST-GL, but not to GSTR3E or GST-R5 ⁄ PTG (data not shown).
A specific and sensitive phosphatase immunoadsorption assay has been developed [34,39], which allows
characterization of the activities of the different glyco1483


S. Munro et al.

Glycogen

Lysate

A

Microsomal

Novel glycogen-targeting subunit of PP1

39
R3E
28

C

0.03

Phosphorylase phosphatase
activity (mU/mg)

Phosphorylase phosphatase
activity (mU/mg)

B

0.02

0.01

0.1

0.075

0.050

0.025

0

0
-RVXF
peptide

+RVXF
peptide

GL

R5

R3E

Fig. 5. Detection of PPP1R3E and its associated phosphatase activity in liver. (A) Rat liver lysate (20 lg protein), microsomal fraction
(20 lg protein) and glycogen fraction (2 lg protein) were subjected
to electrophoresis on 10% SDS ⁄ polyacrylamide gels. After transfer
to nitrocellulose, the blot was probed with 0.5 lgỈmL)1 anti-GSTR3E(1–98). (B) Phosphorylase phosphatase activity associated with
R3E in rat liver lysates (assayed in the presence of 4 nM okadaic
acid). The R3E complex was immunoadsorped from 100 lg of rat
liver lysate. The immune pellets were then assayed for spontaneous phosphorylase phosphatase activity (in the absence of dissociating peptide) and total phosphorylase phosphatase activity
(assayed in the presence of the PP1c-dissociating RVXF containing
peptide). Phosphatase activity is expressed in mmg)1 total protein in the rat liver lysate. The phosphatase activity in control IgG

protein G-Sepharose immune pellets (0.001 mmg)1) was subtracted. Error bars indicate the SEM for assay of three liver lysates,
each assay being performed in triplicate. (C) Comparison of
the total phosphorylase phosphatase activity associated with GL,
R5 and R3E (measured in the presence of the PP1-dissociating
peptide).

gen-targeted forms of PP1. Essentially, using specific
antibodies to a glycogen-targeting subunit of choice, it
is possible to pellet the bound PP1c activity in an
immune complex. However, the interaction of regulatory subunits with PP1c may modify substrate specificity, decreasing the activity of PP1c against some
substrates while increasing it against others. The
immune pellet is therefore assayed for protein phosphatase activity in the absence and presence of a peptide that dissociates the interaction between PP1c and
glycogen-targeting subunits [40]. Inclusion of the dissociating peptide relieves the modification of phosphatase
activity imposed by the glycogen-targeting subunit and
provides a means to calculate the actual amount of
1484

PP1c bound to each subunit. After immunoadsorption
of R3E with anti-R3E(8–23) serum, the spontaneous
phosphorylase phosphatase activity associated with
PPP1R3E (measured in the absence of the dissociating
peptide) was 0.006 ± 0.0008 mmg)1. Addition of
the dissociating peptide to the assay increased the
activity by approximately fourfold to 0.024 ± 0.005
mmg)1. This provided evidence that R3E does
indeed interact with PP1c and suggests that the
interaction of R3E with PP1 inhibits its activity
substantially with phosphorylase a as a substrate
(Fig. 5B). In contrast, the activity of PP1-R3E using
GS as substrate was similar in the presence and

absence of dissociating peptide, demonstrating that
R3E exhibited little or no inhibition of PP1c activity
towards this substrate (Fig. 6B). The glycogen synthase
phosphatase ⁄ phosphorylase phosphatase (GSP ⁄ PhP)
activity ratio for R3E-PP1c of 3.7 is substantially
higher than that calculated for GL (1.9), R5 (0.9) and
R6 ( 2) [34]. Comparison of the level of phosphorylase phosphatase activity associated with PPP1R3E
with that associated with GL and R5 in rat liver,
shows that the activity associated with PPP1R3E is
 30% of that bound to GL, and is slightly higher than
that associated with R5 ⁄ PTG (Fig. 5C).
Effect of induced diabetes and insulin treatment
on the expression and activity of PPP1R3E in vivo
Previous studies [33,34] have shown that streptozotocin-induced diabetes in rats causes 75 and 60% decreases in the hepatic protein phosphatase activity associated with GL and R5 ⁄ PTG, respectively. This response
is accompanied by a corresponding decrease in the
hepatic levels of GL and R5 ⁄ PTG proteins. All of
these effects were restored by the intravenous administration of insulin. The finding that R3E appears to be
most highly expressed in rodent liver prompted investigation into whether this subunit may be regulated
in vivo in liver by streptozotocin-induced diabetes and
changes in insulin levels.
Figure 6(A,B) illustrates the results of assays of antiR3E(8–21)–protein G–Sepharose immunopellets from
liver lysates of control, diabetic and insulin-treated
diabetic rats. The phosphorylase phosphatase and
GSP activities associated with R3E are decreased by
 65–70% in the diabetic rat liver. Furthermore, the
phosphatase activities associated with R3E could be
restored to that of control levels following intravenous
administration of insulin for 96 h. The same percentage decrease in phosphorylase phosphatase and GSP
activities in diabetic livers and restoration by insulin
treatment was observed in the presence of the dissociFEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS

S. Munro et al.

ating peptide (Fig. 6). The activity in IgG control
immune pellets was < 5% of the phosphatase activity
associated with R3E. Analysis of the RNA in the livers
of control and streptozotocin diabetic rats showed that
the R3E mRNA levels varied in parallel with the phosphatase activities of PP1-R3E (Fig. 6C). The data
demonstrate that, like GL and R5 ⁄ PTG, R3E is downregulated in type 1 diabetic animals.

A

0.03

Phosphorylase phosphatase
activity (mU/mg)

-RVXF
+RVXF

0.02

0.01

diabetic +96 h
i nsulin

diabetic

control

0

B
Glycogen synthase phosphatase
activity (mU/mg)

Fig. 6. Effect of streptozotocin-induced diabetes on R3E-associated
phosphorylase and GSP activities and R3E mRNA in rat liver.R3E
immune pellets assayed for phosphorylase phosphatase activity (A)
and GSP (B) activities assayed in the absence and presence of
the PP1c RVXF-containing dissociating peptide. The activities are
expressed as mmg)1 of total protein in the rat liver lysate. Error
bars indicate the SEM. Control rats (n ¼ 3), diabetic rats (n ¼ 5),
diabetic rats +96 h insulin treatment (n ¼ 4).The differences in
spontaneous phosphorylase phosphatase activities (P < 0.01 for
control and diabetic livers, P < 0.001 for diabetic and insulin treated
livers), and the total phosphorylase phosphatase activities in the
presence of the PP1c dissociating peptide (P < 0.02 for control and
diabetic livers, P < 0.001 for diabetic and insulin-treated livers) are
statistically significant. The differences in spontaneous GSP activities (P < 0.05) and total GSP activities in the presence of the
PP1c-dissociating peptide (P < 0.05) are also statistically significant.
(C) Analysis R3E mRNA levels in the livers of control and streptozotocin-induced diabetic rats. The R3E and control b-actin DNA
bands obtained by multiplex RT–PCR using rat R3E-specific and
b-actin-specific primers are stained with ethidium bromide and visualized under UV light.

Novel glycogen-targeting subunit of PP1

0.0004

-RVXF
+RVXF

0.0003

0.0002

0.0001

The novel gene PPP1R3E encoding a putative glycogen-targeting subunit of PP1 is shown here to express
R3E protein in rodent liver. R3E shows <33% amino
acid identity to any of the other glycogen-targeting
subunits, but is highly conserved from rodents to
humans (> 86% identity), suggesting that it may serve
an important nonredundant function. The R3E protein
was found to be present in the hepatic glycogen fraction and to bind to PP1. The phosphorylase phosphatase activity associated with R3E in rat liver was
slightly higher than that bound to R5 ⁄ PTG and
 30% of that bound to the most abundant hepatic
glycogen-targeting subunit GL. However, the GSP ⁄ PhP
activity ratio associated with R3E is 3.7 compared with
1.9 for GL and 0.9 for R5 ⁄ PTG indicating that PP1cR3E has the potential to contribute 60% of the GSP
activity of PP1c-GL in rat liver. The data also indicate
that PP1c-R3E, like PP1c-GL, would be expected to
function mainly as a GSP, whereas PP1c-R5 ⁄ PTG is
more likely to function predominantly as phosphorylase phosphatase.
FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS

C

control

diabetic + 96 h
insulin

Discussion

diabetic

control

0

diabetic

R3E
β-actin

Although analysis of the mRNA encoding rat R3E
revealed that the main tissues of expression are liver
and heart, with only very low levels being present in
skeletal muscle, analysis of the human tissues indicated
that PPP1R3E mRNA is most highly expressed in
skeletal muscle and heart. Very low levels of PPP1R3E
mRNA were detected in most other human tissues
examined, including liver. The difference in tissue distribution between humans and rats reflects in part that
1485


Novel glycogen-targeting subunit of PP1

seen for the GL glycogen-targeting subunit of PP1 [18].
GL, which is highly expressed in rodent liver but only
present at very low levels in rodent skeletal muscle, is
found at appreciable levels in human skeletal muscle
(as well as in human liver). The finding that two glycogen-targeting subunits are highly expressed in human
skeletal muscle while being present at only very low
levels in rodent skeletal muscle may underlie a fundamental difference in the regulation and function of glycogen-bound PP1 in skeletal muscle in humans and
rodents.
The observation that R3E appeared to be predominantly expressed in insulin-sensitive tissues, led to investigation of whether this protein is regulated by insulin
in vivo. Although no evidence was found for acute
regulation via phosphorylase a as seen for PP1-GL,
PP1-R3E associated phosphorylase phosphatase and
GSP activities were substantially decreased in the livers
of diabetic rats and these activities were restored by
insulin treatment. The similar decreases in activity
observed for PP1-GL and PP1-R5 ⁄ PTG in the livers
of diabetic animals was found to correspond to a
decrease in protein and mRNA levels for their glycogen-targeting subunits [34]. Because R3E protein was
barely detectable in liver lysates (Figs 4B and 5A) by
either of two different antibodies, it was not possible
to directly confirm a decrease in R3E protein in the
livers of streptozotocin diabetic rats by immunoblotting. However, examination of R3E mRNA levels
demonstrated a decrease to below detectable levels in
the livers of diabetic rats. It therefore appears that
hepatic R3E, like GL and hepatic R5 ⁄ PTG, is regulated at the transcriptional level by insulin and that R3E
mRNA and consequently protein levels are decreased
in streptozotocin diabetic animals.
The novel PPP1R3G appears to be expressed at low
levels exclusively in brain as judged from mRNA blotting and detection in brain cDNA libraries. This situation is unusual, in that other PP1 glycogen-targeting
subunits are either expressed at low levels ubiquitously

or are present at significant levels in insulin-sensitive
tissues such as liver and skeletal muscle. However, glycogen is a major energy reserve in brain astrocytes and
glycogen mobilization is tightly coupled to neuronal
activity [41].
Conservation of the amino acid sequence of R3G
from human to rodents suggests that, like R3E, it may
perform a distinct and critical function. The generation
of mice lacking the gene encoding the major striated
muscle glycogen-targeting subunit of PP1, GM, has provided evidence to suggest that there is insufficient compensatory response from other subunits because mice
lacking the GM subunit have only 10% muscle glyco1486

S. Munro et al.

gen compared with their wild-type littermates [24,25].
The homozygous deletion of PTG ⁄ R5 ⁄ PPP1R3C has
recently been reported to be embryonic lethal [42]. Mice
heterozygous for this deletion have decreased glycogen
stores and GS activity in muscle, liver and adipose tissue. Glucose intolerance, hyperinsulinaemia and insulin
resistance were also observed to develop with increasing
age. These results indicate that PTG performs a critical
role that cannot be undertaken by the other glycogentargeting subunits. The development of mice lacking
particular subunits may, therefore, uncover whether
there is any functional redundancy among the other
glycogen-targeting subunits of PP1.
The high levels of GL and PPP1R3E mRNA in
human compared with rodent skeletal muscle indicates
that rodents may not be appropriate models from
which to gain an understanding of the hormonal regulation of human skeletal muscle GSP. In addition, this
species-specific difference in the expression of PP1 regulatory subunits is likely to be relevant to the study of
the mechanism of action of insulin on human skeletal

muscle and liver glycogen synthesis and the pathophysiology of human type 2 diabetes.

Materials and methods
Amplification of PPP1R3E and PPP1R3G from
human cDNA libraries
Full-length coding sequences of PPP1R3E and PPP1R3G
were amplified from human brain and testis Matchmaker
cDNA libraries (Clontech, Palo Alto, CA, USA) by two
rounds of PCR using the Advantage GC-cDNA polymerase
and instructions (Clontech). PPP1R3E was amplified by an
initial PCR with the forward primer 1 (nucleotides )105 to
)84) 5¢-GAAGCGGACCCACGGACTTCTG-3¢ and the reverse primer 2 (complementary to nucleotides 957–937 5¢-GA
CTCCCTTGGACCGCTCCCG-3¢), followed by a second
round of PCR with the forward primer 3 (nucleotides 1–21
5¢-ATGTCCGCTGAGCGGCCCCCG-3¢) and the reverse
primer 4 (complementary to nucleotides 837–815 5¢-GATA
AAGTGGATCCAGCCCCATAGGGGCGCGG-3¢) and the
reverse primer 8 (complementary to nucleotides 1074–1058
5¢-GAGCGCGTCCGCAGGGCACGC-3¢). PCR products
were resolved on 1% (w ⁄ v) agarose gels, gel-purified,
cloned into pCR2.1 TOPO vector (Invitrogen, Carlsbad,
CA, USA) and sequenced in both directions using M13 forward and reverse primers. DNA sequencing was performed
in conjunction with the Sequencing Service managed by
Dr Nick Helps (School of Life Sciences, University of
Dundee; ) using an Applied
Biosystems 373 A DNA sequencer or Big-Dye Ver 3.1
chemistry on an Applied Biosystems model 3730 automated
capillary DNA sequencer.

FEBS Journal 272 (2005) 1478–1489 ª 2005 FEBS

S. Munro et al.

RNA analyses
Northern blots (Clontech) contained  2 lg poly(A)+ RNA
from different tissues of fed rats post mortem and human tissues collected from fed individuals no more than 3 h after
death. Blots were hybridized a32P-labelled cDNA probes
according to the manufacturer’s instructions with the final
wash in 10 mmolỈL)1 NaCl, 1.5 mmolỈL)1 sodium citrate,
0.1% SDS, at 55 °C. Levels of R3E and control b-actin mRNA
transcripts were assessed in total rat liver RNA by multiplex
RT-PCR (Promega, Madison, WI, USA) as described previously [34]. The rat R3E specific forward and reverse primers
were 5¢-ATGTCCCGTGAGCGGCCCCCG-3¢ and 5¢-GAT
AAAGTGGATCCAGCCCTGCG-3¢, respectively.

Treatment of animals
Diabetes was induced with either intravenous or intraperitoneal injection of streptozotocin into male Wistar rats
and insulin was subsequently administered intravenously
into some of the rats for 96 h [34]. Blood glucose levels
were elevated ‡ fourfold in diabetic animals prior to insulin treatment. The rats were killed by suffocation in CO2
and tissues were excised, freeze-clamped, and stored at
)80 °C. All procedures were performed in accordance with
the guidelines of the ethical committees of the University
of Dundee or the Katholieke Universiteit Leuven.

Immunological techniques
Homogenization of tissues was performed as detailed in
Munro et al. [18]. Homogenates were centrifuged at 16 000 g
for 10 min, and the supernatants were snap-frozen in liquid

nitrogen and stored at )80 °C. Preparation of subcellular
fractions was performed as detailed in Browne et al. [34].
Proteins were separated by 10% SDS ⁄ PAGE, transferred to
nitrocellulose, and probed with affinity purified antibodies.
Peptides were synthesized by G. Bloomberg (University of
Bristol, UK); antibodies were raised in sheep by Diagnostics
Scotland (Penicuik, Midlothian, UK) and affinity purified in
conjunction with the Division of Signal Transduction Therapy, University of Dundee coordinated by H. McLauchlan
and J. Hastie. Antibodies to human PP1b peptide (amino
acids 316–327) and human PPP1R3E(8–23) were affinity
purified against their respective peptides. Antibodies to
human GST-PPP1R3E(1–98) were affinity purified against
MBP-PPP1R3E. Immunoblotting followed by detection of
immunoreactive bands by enhanced chemiluminescence was
performed as described in Munro et al. [18].

Protein phosphatase assays
PP1 activities were determined by release of [32P]phosphate
from phosphorylase a (10 mmolặL)1, phosphorylated by

FEBS Journal 272 (2005) 14781489 ê 2005 FEBS

Novel glycogen-targeting subunit of PP1

phosphorylase kinase) and GS (1 mmolỈL)1, phosphorylated by GSK3) in the presence of 4 nm okadaic acid for
10 min at 30 °C. For immunoadsorption of PP1-GL,
PPP1R5 and PPP1R3E with anti-GL, anti-R5 and antiR3E sera, respectively, lysates were prepared in the presence of 100 nm okadaic acid. Immune pellets from 100 lg
of liver lysate were washed five times in the presence of
4 nmolỈL)1 okadaic acid, and PP1 activities in the immune
pellets were assayed as described above either before

(‘spontaneous’ activity) or after (‘total’ activity) preincubation with 0.1 mgỈmL)1 ‘dissociating’ peptide (GKRTNLR
KTGSERIAHGMRVKFNPLALLLDSC) that causes the
release of free PP1c from the glycogen-targeting subunit
[34,39]. One unit of activity is the amount of enzyme that
catalyses the release of 1 mmol of [32P]phosphate
per minute. Statistical significance was assessed using the
Student’s t-test.

Acknowledgements
The work was supported by the UK Medical Research
Council, UK and Diabetes UK. SM was initially the
recipient of a Cooperative Awards in Science and
Engineering postgraduate studentship from the Biotechnology and Biological Research Sciences Council,
UK and Novo Nordisk, Bagsvaerd, Denmark. Subsequently, SM was supported on a postdoctoral research
assistantship by Diabetes UK. HC is a postdoctoral
fellow of the Fund for Scientific Research-Flanders.

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