Insights into Lafora disease: Malin is an E3 ubiquitin
ligase that ubiquitinates and promotes the
degradation of laforin
Matthew S. Gentry, Carolyn A. Worby, and Jack E. Dixon*
Departments of Pharmacology and Cellular and Molecular Medicine, School of Medicine, and Department of Chemistry and Biochemistry,
University of California at San Diego, La Jolla, CA 92093
Contributed by Jack E. Dixon, April 25, 2005
Lafora disease (LD) is a fatal form of progressive myoclonus
epilepsy caused by recessive mutations in either a gene encoding
a dual-specificity phosphatase, known as laforin, or a recently
identified gene encoding the protein known as malin. Here, we
demonstrate that malin is a single subunit E3 ubiquitin (Ub) ligase
and that its RING domain is necessary and sufficient to mediate
ubiquitination. Additionally, malin interacts with and polyubiq-
uitinates laforin, leading to its degradation. Missense mutations in
malin that are present in LD patients abolish its ability to polyu-
biquitinate and signal the degradation of laforin. Our results
demonstrate that laforin is a physiologic substrate of malin, and
we propose possible models to explain how recessive mutations in
either malin or laforin result in LD. Furthermore, these data
distinguish malin as an E3 Ub ligase whose activity is necessary to
prevent a neurodegenerative disease that involves formation of
nonproteinacious inclusion bodies.
progressive myoclonus epilepsy ? phosphatase ? neurodegenerative disease
seizures, and progressive neurologic dysfunction. The five most
common forms of PME, Unverricht–Lundborg disease, Lafora
disease (LD), myoclonic epilepsy with ragged red fibers, the
neuronal ceroid lipofuscinosis, and type I sialidosis, differ in the
severity of their clinical features, molecular origins, pathogen-
esis, and prognosis (1). Although the mutated gene or genes are
PMEs, their molecular etiologies remain elusive.
LD (OMIM 254780) is an autosomal recessive progressive
myoclonus epilepsy that was first described in 1911 (2–5). LD
commonly presents in the first or second decade of life with
epileptic seizures followed by progressive central nervous system
degeneration beginning with myoclonic seizures, tonic-clonic
seizures, focal occipital seizures, intellectual decline, severe
motor and coordination deterioration, constant myoclonus, and,
finally, death within 10 years of onset (4–7). A hallmark of the
disease is the presence of periodic acid-Schiff-positive polyglu-
cytoplasm (5, 8). LBs are pathognomic of LD and may be the
causative agent of LD neurodegeneration. The inclusion bodies
appear to arise from an undefined defect or defects in glycogen
metabolism (e.g., misregulated glycogen synthesis or degrada-
tion). LBs are found in most organs and are prominent in
neuronal perikarya, the liver, and muscle (5, 9–11).
Two groups independently identified EPM2A (epilepsy of pro-
Mutations in EPM2A are responsible for ?48% of LD cases (14).
Of the remaining cases, ?40% are the result of mutations in
EPM2B (14, 15). To date, the molecular mechanisms underlying
LD in patients harboring mutations in either EPM2A or EPM2B
have not been defined.
The human, mouse, and rat orthologs of EPM2A are 94%
similar at the amino acid level (16). EPM2A encodes a protein
he progressive myoclonus epilepsies (PMEs) share a char-
acteristic triad of features: myoclonic seizures, tonic-clonic
product of 331 aa, named laforin, that contains the dual-
specificity protein phosphatase (DSP) active site motif,
HCXXGXXRS?T (Fig. 1A) (12, 13, 17, 18). Accordingly, recom-
binant laforin can hydrolyze phosphotyrosine and phospho-
serine?threonine substrates in vitro (16, 19). The amino terminus
of laforin contains a carbohydrate-binding domain (CBD) that
targets it to subcellular sites of glycogen synthesis and promotes
laforin’s binding to glycogen in vitro and in vivo (Fig. 1A) (19).
Laforin preferentially binds LBs over glycogen in vitro and
colocalizes with LBs in both a transgenic mouse model and in
to the endoplasmic reticulum and colocalizes with glycogen
synthase (16, 20–22). Most of the reported missense mutations
profoundly affect laforin’s phosphatase activity and?or its ability
to bind glycogen (19, 20, 23, 24). The exception is the G240S
mutation, which has WT protein tyrosine phosphatase activity
and binds glycogen but has reduced interaction with PTG?R5,
one of the glycogen-targeting regulatory subunits of protein
phosphatase 1 (24). Cumulatively, these data place laforin in the
context of a multiprotein complex associated with intracellular
glycogen particles and suggest that laforin is involved in the
regulation of glycogen metabolism, either by promoting proper
glycogen production and?or by removal of aberrant glycogen.
However, the mechanism explaining laforin’s role in the molec-
ular etiology of LD is unknown.
The second LD gene, EPM2B, encodes a 395-aa protein called
malin (14). Malin contains a consensus RING-HC domain and six
NHL domains (Fig. 1B) (14). RING domains are characteristic of
one class of E3 ubiquitin (Ub) ligases (25, 26). Modification of
is activated by and transferred from the activating enzyme (E1) to
a conjugating enzyme (E2) and finally to a substrate with the
involvement of an Ub ligase (E3) (27). Ubiquitination of a protein
ubiquitination can change the activity, binding capacity, or local-
to WD40 repeats (30, 31). Although overexpressed malin colocal-
izes with laforin at the endoplasmic reticulum (14), currently no
molecular mechanism connects malin with LD.
Herein, we demonstrate that malin’s RING domain possesses in
vitro E3 Ub ligase activity. This activity depends on one of four E2
enzymes, UbcH2, UbcH5a, UbcH5c, or UbcH6. Using a yeast
two-hybrid screen, we identified laforin as a binding partner of
malin and present evidence that malin directly binds laforin in vitro
Abbreviations: LD, Lafora disease; LB, Lafora body; HA, hemagglutinin; IP, immunopre-
cipitation; CBD, carbohydrate-binding domain; DSP, dual-specificity protein phosphatase;
ubiquitin; biotin-Ub, biotinylated Ub; WCL, whole-cell lysate.
*To whom correspondence should be addressed at: Department of Pharmacology, Univer-
sity of California at San Diego School of Medicine, Leichtag Building, Room 284, 9500
Gilman Drive, La Jolla, CA 92093-0721. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
June 14, 2005 ?
vol. 102 ?
no. 24 ?
and interacts with laforin in vivo. Additionally, we show that laforin
is polyubiquitinated in a malin-dependent manner in vitro and in
vivo and that this polyubiquitination leads to laforin degradation in
vivo. LD mutations in malin abolish both laforin’s polyubiquitina-
tion and its degradation. Thus, malin regulates the protein concen-
We discuss how these results are in agreement with both clinical
immunohistochemistry data and the genetics of LD.
Plasmids and Proteins. Plasmids encoding human malin were con-
structed by PCR using Open Biosystems clone CA450023 and a
was inserted into pcDNA3.1NF (32), pET-GSTX (33), pET21a
(Novagen), and pGBKT7 (Clontech). Mutations were introduced
by using the QuikChange mutagenesis kit (Stratagene). HA3-Ub
San Francisco). Hemagglutinin (HA)-Ub, HA-SUMO, pET21a
laforin-His6, and pcDNA3.1NF-laforin are described in refs. 19
Recombinant malin and laforin were purified from soluble
bacterial lysates by using Ni-nitrilotriacetic acid (NTA) agarose or
successive Ni-NTA agarose and glutathione-agarose affinity chro-
details are provided in Supporting Text, which is published as
supporting information on the PNAS web site. WT ubiquitin,
biotinylated Ub (biotin-Ub), human E1, and human E2 enzymes
were purchased from Boston Biochem (Cambridge, MA).
Cell Culture and Transfection. Adenovirus-transformed human em-
CO2 in DMEM (Invitrogen) supplemented with 10% FBS?50
units/ml penicillin/streptomycin?4 mM glutamine. Subconfluent
cultures of HEK293T cells (2 ? 106cells per 100-mm dish) were
Science, Indianapolis) according to the manufacturer’s protocol
and were cultured for 24 h to allow protein expression.
Antibodies and Immunoprecipitations (IPs). IPs and denaturing IPs
were essentially performed as described in refs. 36 and 37. Specific
details are available in Supporting Text. Western blots were probed
with one of the following antibodies: mouse anti-Ub (Covance,
Richmond, CA), biotin-horseradish peroxidase (HRP), (Boston
Biochem), mouse anti-His-HRP (Santa Cruz Biotechnology),
mouse anti-myc-HRP (Roche Molecular Biochemicals), mouse
anti-FLAG-HRP (Sigma), or rat anti-HA (Roche Molecular Bio-
chemicals). Goat anti-rat-HRP (Roche Molecular Biochemicals),
goat anti-mouse-HRP (Amersham Pharmacia), and rabbit anti-
mouse (Amersham Pharmacia) secondary antibodies were used
when needed. The HRP signal was detected by using SuperSignal
West Pico or Femto (Pierce). Some blots were stripped with
Yeast Two-Hybrid Screen. The bait plasmid, pGBKT7-malin
(pMG101), was constructed by subcloning full-length malin into
pGBKT7 (Clontech). pGBKT7-RING (pMG106) contained ma-
lin’s amino terminus through Asn-128, pGBKT7–6NHL contained
malin’s carboxyl terminus from Cys-78, pACTII-CBD contained
laforin’s amino terminus through Gly-120, and pACTII-DSP con-
tained laforin’s carboxyl terminus from Leu-161. All of the two-
hybrid clones (including the pGBKT7-malin point mutants) ex-
by Western blotting. pGBKT7–6NHL expression was 2- to 3-fold
lower. The yeast two-hybrid screen was performed per the manu-
facturer’s instructions; specific details are in Supporting Text.
In Vitro Binding Assay. Empty Ni-NTA agarose (Qiagen, Valencia,
laforin-His6was incubated in buffer A (50 mM Tris, pH 8.0?300
mM NaCl?20 mM imidazole, pH 8.0?15% maltose?0.05% 2-mer-
captoethanol?protease inhibitors) at 4°C for 2 h with35S-labeled
FLAG-malin that was made in vitro by using the TNT T7-coupled
reticulocyte lysate system (Promega). The agarose was washed two
times with buffer A and two times in buffer B (50 mM Tris, pH
8.0?600 mM NaCl?20 mM imidazole, pH 8.0?15% maltose?0.05%
2-mercaptoethanol?protease inhibitors). Proteins were eluted at
95°C in NuPage buffer, separated by SDS?PAGE, transferred to
nitrocellulose, and analyzed to detect laforin-His6and35S-malin.
In Vitro Ubiquitination Assay. The in vitro ubiquitination assay was
performed essentially as described in ref. 38; specific details are
provided in Supporting Text. When laforin was used in this assay,
35S-labeled FLAG-laforin was made in vitro by using the TNT
T7-coupled reticulocyte lysate system (Promega); it was then
immunoprecipitated out of the reaction mixture, washed three
times, and eluted into 50 ?l of Ub assay buffer with 10 ?g of FLAG
Malin Functions as an E3 Ubiquitin Ligase in Vitro. Malin contains a
RING-HC-type zinc finger and six NHL domains (14) (Fig. 1B).
Based on subclassification of RING domains (39), malin falls into
the RING-HCa family. The presence of a RING finger suggests
showed that only 60% of RING-HCa domain proteins exhibited in
vitro E3 Ub ligase activity (39).
We therefore first tested whether malin possesses E3 ligase
activity in an in vitro Ub assay (38, 40, 41). Previous work has
demonstrated that E3 ligases can polyubiquitinate artificial protein
substrates, often GST, in an in vitro reaction mixture (40). Recom-
binant, affinity-purified GST-malin-His6 was combined with a
recombinant human E1 enzyme, 1 of 10 different recombinant
human E2 enzymes, ATP, and biotin-Ub. Ubiquitinated species
were monitored by Western blot analysis using avidin-HRP to
detect biotin-Ub. Robust malin-dependent ubiquitination was con-
sistently detected when one of four E2 enzymes was present:
UbcH2, UbcH5a, UbcH5c, or UbcH6 (Fig. 2A, lanes 4, 8, 12, and
14). This ubiquitination was not dependent on the presence of
malin’s GST tag because malin-His6also provided robust E3 ligase
activity (data not shown). No ubiquitination was detected when
bacterially purified GST was added to the reaction in place of
GST-malin-His6(Fig. 2A, compare lanes 3 and 4, 7 and 8, 11 and
12, and 13 and 14). The substrates ubiquitinated in this assay were
largely the GST-E2 enzymes and not autoubiquitinated malin,
detected the same laddered, high molecular mass bands (data not
protein, with missense mutations in red and other mutations in black. (A)
Laforin contains a CBD and a DSP domain. (B) Malin contains a RING domain
and six NHL repeats.
A schematic of laforin and malin. LD mutations are shown for each
www.pnas.org?cgi?doi?10.1073?pnas.0503285102Gentry et al.
shown). Because RING domains are often sufficient to support
E3 activity. Indeed, ubiquitination depended on malin’s RING
domain but not on its NHL domains (Fig. 6A, which is published
as supporting information on the PNAS web site). Additionally,
ubiquitination was also dependent on the presence of all of the in
vitro assay components (Fig. 6B). These results demonstrate that
malin functions as an E2-dependent E3 Ub ligase in vitro.
Malin Interacts with Laforin. To further understand malin’s role in
LD, we sought to identify malin’s endogenous substrate(s). We
performed a yeast two-hybrid screen using a human brain cDNA
library and identified several clones that interacted with either
malin or malin’s NHL domains (data not shown). Plasmid rescue
and sequencing identified three clones as EPM2A, the gene encod-
To map the domains responsible for the malin–laforin interac-
tion, we generated two-hybrid clones containing malin’s RING or
NHL domain and tested the ability of these constructs to interact
with laforin’s CBD or DSP domain (Fig. 1). Malin’s NHL domain
interacted with laforin as strongly as full-length malin, whereas the
only full-length laforin interacted with malin (Fig. 3A). We then
tested the effect of various LD missense mutations on the malin–
laforin interaction. RING domain mutations (C26S and P69A) did
not decrease the malin–laforin interaction, but NHL domain mu-
tations (E280K and Q302P) abolished the interaction (Fig. 3B,
compare 4 and 5 with 6 and 7). Thus, malin’s six NHL domains
likely function as a substrate-interacting motif, whereas malin’s
RING domain provides E3 Ub ligase activity. These data suggest
that laforin could potentially serve as a substrate for malin. In
addition, our results suggest that mutations in malin found in LD
by disrupting malin’s substrate binding domain or its E2 binding
Single subunit E3 ligases often interact directly with their sub-
strates, whereas multisubunit E3 ligases require protein complexes
to interact with substrate(s) (42, 43). To determine whether malin
would behave as a single subunit E3 ligase,35S-labeled in vitro
translated malin was immunopurified from the reticulocyte extract
and added to Ni-NTA agarose (control) or recombinant laforin-
His6immobilized on Ni-NTA agarose. Malin strongly associated
with laforin without the aid of an adaptor protein, thus functioning
like a single subunit E3 ligase (Fig. 3C).
To determine whether this interaction could be maintained in
vivo, we coexpressed FLAG-tagged laforin and myc-tagged
malin in HEK293T cells. Laforin coimmunoprecipitated with
malin (Fig. 3D Upper, lane 1); it coimmunoprecipitated to a
lesser degree with malin RING domain mutants (Fig. 3D Upper,
compare lane 1 with lanes 3–5) and to a still lesser degree with
malin NHL domain mutants (Fig. 3D Upper, compare lane 1 and
lanes 6 and 7). Similar results were obtained by immunoprecipi-
tating laforin and immunobloting for malin (data not shown).
Although the NHL mutations decreased the malin–laforin in-
teraction, they did not fully disrupt the interaction, as was
observed by means of the yeast two-hybrid assay. Although we
do not have a definite answer for these observations, the
difference could result from a posttranslational modification or
an additional interacting protein or proteins that may strengthen
the interaction in mammalian cells. Strikingly, we noticed that
there was much less total laforin in whole-cell lysate (WCL)
when WT malin was present compared with empty vector or
mutant malin (Fig. 3D Lower, top panel, compare lane 1 and
performed as described in Methods with E1 enzyme, 1 of 10 E2 enzymes, ATP,
biotin-Ub, and recombinant GST-malin-H6(?) or GST alone (?).
Malin provides E3 Ub ligase activity in vitro. In vitro Ub assays were
with the various plasmid combinations, and the activation of the HIS and ADE
reporters was assessed by growth on selective plates. (C)35S-labeled, in vitro
translated malin (35S-malin) was incubated with Ni-NTA agarose or Ni-NTA
agarose bound to recombinant laforin-H6. The agarose was washed exten-
sively, and bound proteins were analyzed by means of Western blotting with
input and bound (pulldown)35S-malin were exposed for different lengths of
time. (D) HEK293T cells were cotransfected with 5 ?g of malin-myc, empty
vector, or mutant malin-myc and 5 ?g of FLAG-laforin. WCLs were immuno-
blotted with ?-myc and reprobed with ?-FLAG. The nitrocellulose from the
subjected to IP with ?-myc followed by ?-myc immunoblotting and reprobed
Malin interacts with laforin. (A and B) Yeast cells were transformed
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lanes 2–7). The decreased overall amount of laforin in the WCL
suggests that the malin–laforin interaction is quite robust. These
data also show that laforin interacts with malin in vivo and
suggest that malin is likely involved in regulating laforin protein
Laforin Is Ubiquitinated in a Malin-Dependent Manner. Because
laforin levels are greatly decreased in cells expressing malin, we
investigated whether malin might be responsible for regulating
laforin levels by means of polyubiquitination and subsequent deg-
radation. To determine whether malin was able to promote the
ubiquitination of laforin in vivo, FLAG-tagged laforin was cotrans-
fected with WT, mutant RING, or mutant NHL forms of malin in
HEK293T cells. Of the three forms of malin, only WT malin
reduced laforin protein levels (Fig. 4A Left Top, lanes 1 and 8 vs.
lanes 2–7). Similarly, multiple Ub species coimmunoprecipitated
with laforin only in cells coexpressing WT malin (Fig. 4A Right
Bottom, lanes 1 and 8 vs. lanes 2–7). The Ub species that coimmu-
noprecipitated with laforin corresponded to ?8-kDa increases in
kDa, ?8 kDa larger than laforin. A longer exposure of the
immunoprecipitated anti-FLAG blot revealed higher molecular
mass versions of laforin when malin was present, again at ?8-kDa
RING or NHL domains abolished the reduction in laforin protein
levels, the modified forms of laforin, and the coimmunoprecipitat-
ing Ub species (Fig. 4A). These data demonstrate that laforin is
ubiquitinated and potentially targeted for degradation in a malin-
Phosphorylation often regulates ubiquitination, either positively
or negatively (44–46). Because laforin is a phosphatase, we tested
whether laforin’s phosphatase activity was required for its ubiquiti-
nation. LaforinC266Slacks phosphatase activity (19), but it was still
ubiquitinated and degraded in a malin-dependent manner (Fig. 4A
Left and Right, lane 8). Thus, laforin’s phosphatase activity is not
required for its ubiquitination or degradation.
To determine the specificity of malin’s ligase activity, we also
to laforin in a malin-dependent manner. Only HA-Ub coimmuno-
precipitated with laforin in a malin-dependent manner (Fig. 7 Top,
which is published as supporting information on the PNAS web
site). In addition, laforin was still modified in cells overexpressing
malin and HA-SUMO by the endogenous Ub (Fig. 7 Bottom),
showing that SUMO is not transferred to laforin.
Because mono-?di-ubiquitination and polyubiquitination signal
very different fates for an ubiquitinated protein, we wanted to
determine the ubiquitination status of laforin. To determine
whether laforin had multiple Ubs covalently attached to it, we
coexpressed FLAG-laforin and malin-myc in HEK293T cells. IP
with anti-FLAG antibody and immunoblotting with anti-FLAG
and anti-Ub detected up to six forms of laforin, including five
modified forms that comigrated with Ub (Fig. 4B). Additionally,
was stained with Ponceau S to monitor loading. Additionally, WCLs were subjected to IP with ?-FLAG followed by immunoblotting with ?-FLAG and reprobed
subjected to IP with ?-FLAG, immunoblotted with ?-FLAG, and reprobed with ?-Ub. (C) HEK293T cells were cotransfected with malin-myc, FLAG-laforin, HA-Ub,
and HA-Ub lacking lysines (HA-Ubk-less) as indicated. WCLs were subjected to IP with ?-FLAG, immunoblotted with ?-FLAG, and reprobed with ?-HA.
Laforin is polyubiquitinated in a WT malin-dependent manner. (A) HEK293T cells were cotransfected with 5 ?g of malin-myc, empty vector, or mutant
www.pnas.org?cgi?doi?10.1073?pnas.0503285102Gentry et al.
the Ub ladder coimmunoprecipitated with laforin after lysing cells
in buffer containing 2% SDS and denaturing proteins by heating
the lysate at 95°C for 10 min (data not shown). This treatment
should break all noncovalent protein–protein interactions. These
Ub molecules in the presence of malin.
Multiple Ub attachment could occur by means of two different
mechanisms: multiple single Ubs attached to multiple lysines
(multiubiquitination) or a chain of Ubs attached to a single lysine
(polyubiquitination). These two types of ubiquitination trigger
different outcomes for a target protein. To determine whether
laforin was multiubiquitinated or polyubiquitinated, we expressed
FLAG-laforin, malin-myc, and either HA-Ub or HA-Ub-lacking
lysines (HA-Ubk-less) in HEK293T cells. The Ub lacking all lysines
is unable to form polyubiquitin chains and thus can only be
efficiently involved in multiubiquitination. Laforin was ubiquiti-
nated by HA-Ub but not by HA-Ubk-less(Fig. 4C), demonstrating
that laforin is polyubiquitinated in a malin-dependent manner.
Malin Polyubiquitinates Laforin and Promotes Its Degradation. To
again used the same in vitro ubiquitination assay previously de-
scribed but supplemented the reactions with immunopurified35S-
labeled in vitro translated laforin. After performing the reaction at
eliminate all other ubiquitinated proteins in the mixture (specifi-
cally, the GST-E2s). Ubiquitination of35S-laforin was evaluated by
Western blot analysis of biotin-Ub and autoradiography. High
seen only in the presence of bacterially expressed GST-malin-His6
and all other reaction components (Fig. 5A). These data provide
direct evidence that malin induces polyubiquitination of laforin in
vitro and that malin does not need additional cellular components
to polyubiquitinate laforin.
To prove that laforin polyubiquitination triggers its degradation,
we coexpressed FLAG-laforin with either malin-myc or malinC26S-
myc in HEK293T cells. MalinC26Sis a LD mutation that disrupts
malin’s RING domain and decreases malin’s in vitro E3 activity
(data not shown), likely by disrupting malin’s interaction with E2
enzymes. Laforin protein levels were decreased in a malin dose-
dependent manner (Fig. 5B Left). Alternatively, laforin protein
levels were unchanged with increasing amounts of malinC26S-myc
levels is not due to a transcription?translation artifact but to the
show that laforin is polyubiquitinated by malin and targeted for
In this study, we show that malin is a single-subunit, E2-dependent
E3 Ub ligase and that it interacts with laforin both in vitro and in
vivo. Missense mutations in malin’s NHL domains found in LD
patients diminish this interaction. Furthermore, malin polyubiq-
uitinates laforin, and this polyubiquitination promotes laforin’s
degradation. Mutations in malin found in LD patients abolish both
laforin’s polyubiquitination and its degradation. Thus, malin regu-
lates laforin protein concentrations by means of polyubiquitin-
be abrogated by means of mutations that disrupt its ability to bind
either an E2 enzyme or its substrate(s). This assertion is supported
by the location of missense mutations within the malin protein in
LD patients. The mutations fall in malin’s RING and NHL
domains. The only missense mutation not located in one of these
suggest that this residue is likely involved in malin’s ability to bind
an E2 enzyme (47–49). These data, together with our findings,
onset of LD in patients with EPM2B mutations.
There is indirect evidence suggesting that laforin is involved in
proper glycogen anabolism. Laforin binds R5?PTG, a regulatory
subunit of protein phosphatase 1 that is a critical positive regulator
of glycogen synthesis (24, 50). R5?PTG directly binds laforin,
glycogen synthase, phosphorylase, and phosphorylase kinase, act-
ing as a molecular scaffold by assembling the glycogen machinery
(24, 50, 51). Additionally, these proteins are likely to compete for
binding to the PTG scaffold (24, 52). Therefore, laforin could
promote proper glycogen anabolism by dephosphorylating a nec-
essary component of glycogen synthesis and?or by competing for
binding on R5?PTG.
Alternatively, recent studies suggest that laforin may act as a
regulator of aberrant glycogen to inhibit its accumulation. This
model postulates that laforin recognizes LBs, by means of its CBD,
and initiates mechanisms to inhibit LB formation and?or promote
LB destruction through its phosphatase domain (20, 21). Multiple
groups have shown that laforin preferentially binds LBs or LB-like
material over glycogen both in vitro and in vivo (20, 21, 53). Thus,
it is possible that laforin’s role in suppressing LD begins after LB
formation by inhibiting the formation, or by clearance and?or
degradation, of LBs.
Interestingly, Chan et al. (21) reported that they were unable to
detect endogenous laforin in tissue from non-LD patients by using
multiple polyclonal ?-laforin antibodies, but they could detect
endogenous laforin in tissue from LD patients carrying mutations
which demonstrate that malin promotes laforin degradation. Col-
lectively, these clinical and biochemical data demonstrate that
malin regulates the concentration of laforin in situ by means of
Ub-dependent degradation and that loss-of-function malin muta-
tions cause laforin levels to increase. However, if laforin degrada-
vitro ubiquitination assays were performed as described in Fig. 2 but were
supplemented with35S-labeled, in vitro translated laforin (35S-laforin).35S-
detected with avidin-HRP. (B) HEK293T cells were cotransfected with increas-
ing micrograms (0–5) of malin-myc or mutant malin-myc (C26S-myc), 5 ?g of
FLAG-laforin, and enough empty vector to make the total transfection
amount 10 ?g. WCLs were immunoblotted with ?-FLAG and reprobed with
?-myc. The nitrocellulose was stained with Ponceau S to monitor loading.
Malin polyubiquitinates laforin and promotes its degradation. (A) In
Gentry et al.
June 14, 2005 ?
vol. 102 ?
no. 24 ?
tion is malin’s only LD-linked function, then these results appear to Download full-text
be in conflict with the genetics of LD, because recessive mutations
in either laforin or malin cause LD. We envision two scenarios that
explain these data. The models are valid if malin and laforin are
involved in proper glycogen anabolism or aberrant glycogen catab-
olism (e.g., LB degradation?clearance).
X, and it is the accumulation of protein X that causes LD when
malin is mutated. Malin’s regulation of laforin concentrations
would be a secondary regulatory function of malin, and increases
in laforin concentrations would not be the cause of LD in patients
with mutations in malin. Because phosphorylation often regulates
this dephosphorylation event would trigger protein X to be ubi-
quitinated by malin and degraded. Once protein X is degraded,
that laforin and malin function on the same substrate (protein X)
and promote its degradation.
Alternatively, laforin could be both an activator and repressor of
proper glycogen metabolism. In this model, laforin’s CBD would
function to localize laforin and allow laforin to dephosphorylate
substrate X; after dephosphorylation, the next event, either proper
glycogen formation or inhibition of aberrant glycogen accumula-
tion, would not occur until laforin is polyubiquitinated and de-
graded. In this model, laforin is analogous to previously postulated
The activators function to assemble the necessary reagents for
transcription, but their degradation is required to enable RNA
polymerase to leave the promoter and transcribe downstream
of LD in patients with mutations in malin. Patients with mutations
in malin would accumulate laforin and develop LD because de-
Patients with mutations in laforin would develop LD because
laforin would be unable to dephosphorylate a necessary substrate
in glycogen metabolism.
degradation, coupled with the clinical immunohistochemistry data
from affected and unaffected patient tissue (21), suggests that
However, both models should be explored in detail to determine
which provides the best molecular explanation of LD.
We thank Vishva Dixit for various Ub plasmids, Mike Begley for
assistance with the in vitro binding assay, and Dr. Gregory S. Taylor and
David J. Pagliarini for insightful discussions. This work was supported by
National Institutes of Health (NIH)?National Cancer Institute Grant
Cancer Institute (J.E.D.).
1. Lehesjoki, A. E. (2002) Adv. Neurol. 89, 193–197.
2. Van Heycop Ten Ham, M. W. (1975) in Handbook of Clinical Neurology, eds. Vinken,
P. J. & Bruyn, G. W. (North–Holland, Amsterdam), Vol. 15, pp. 382–422.
3. Berkovic, S. F., Andermann, F., Carpenter, S. & Wolfe, L. S. (1986) N. Engl. J. Med.
4. Minassian, B. A. (2002) Adv. Neurol. 89, 199–210.
5. Lafora, G. A. B. G. (1911) Z. Ges. Neurol. Psychiatr. 6, 1–14.
6. Berkovic, S. F., So, N. K. & Andermann, F. (1991) J. Clin. Neurophysiol. 8, 261–274.
7. Berkovic, S. F., Cochius, J., Andermann, E. & Andermann, F. (1993) Epilepsia 34,
Suppl. 3, S19–S30.
8. Collins, G. H., Cowden, R. R. & Nevis, A. H. (1968) Arch. Pathol. 86, 239–254.
9. Harriman, D. G., Millar, J. H. & Stevenson, A. C. (1955) Brain 78, 325–349.
10. Schwarz, G. A. & Yanoff, M. (1965) Arch. Neurol. 12, 172–188.
11. Carpenter, S. & Karpati, G. (1981) Neurology 31, 1564–1568.
12. Minassian, B. A., Lee, J. R., Herbrick, J. A., Huizenga, J., Soder, S., Mungall, A. J.,
Dunham, I., Gardner, R., Fong, C. Y., Carpenter, S., et al. (1998) Nat. Genet. 20,
13. Serratosa, J. M., Gomez-Garre, P., Gallardo, M. E., Anta, B., de Bernabe, D. B.,
Lindhout, D., Augustijn, P. B., Tassinari, C. A., Malafosse, R. M., Topcu, M., et al.
(1999) Hum. Mol. Genet. 8, 345–352.
14. Chan, E. M., Young, E. J., Ianzano, L., Munteanu, I., Zhao, X., Christopoulos, C. C.,
Avanzini, G., Elia, M., Ackerley, C. A., Jovic, N. J., et al. (2003) Nat. Genet. 35,
15. Chan, E. M., Omer, S., Ahmed, M., Bridges, L. R., Bennett, C., Scherer, S. W. &
Minassian, B. A. (2004) Neurology 63, 565–567.
16. Ganesh, S., Agarwala, K. L., Ueda, K., Akagi, T., Shoda, K., Usui, T., Hashikawa, T.,
Osada, H., Delgado-Escueta, A. V. & Yamakawa, K. (2000) Hum. Mol. Genet. 9,
17. Yuvaniyama, J., Denu, J. M., Dixon, J. E. & Saper, M. A. (1996) Science 272,
18. Maehama, T. & Dixon, J. E. (1998) J. Biol. Chem. 273, 13375–13378.
19. Wang, J., Stuckey, J. A., Wishart, M. J. & Dixon, J. E. (2002) J. Biol. Chem. 277,
& Yamakawa, K. (2004) Biochem. Biophys. Res. Commun. 313, 1101–1109.
21. Chan, E. M., Ackerley, C. A., Lohi, H., Ianzano, L., Cortez, M. A., Shannon, P.,
Scherer, S. W. & Minassian, B. A. (2004) Hum. Mol. Genet. 13, 1117–1129.
& Scherer, S. W. (2001) Ann. Neurol. 49, 271–275.
Scherer, S. W. & Minassian, B. A. (2004) Hum. Mutat. 23, 170–176.
24. Fernandez-Sanchez, M. E., Criado-Garcia, O., Heath, K. E., Garcia-Fojeda, B.,
Medrano-Fernandez, I., Gomez-Garre, P., Sanz, P., Serratosa, J. M. & Rodriguez de
Cordoba, S. (2003) Hum. Mol. Genet. 12, 3161–3171.
25. Pickart, C. M. (2001) Annu. Rev. Biochem. 70, 503–533.
26. Hershko, A. & Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425–479.
27. Hershko, A., Heller, H., Elias, S. & Ciechanover, A. (1983) J. Biol. Chem. 258,
28. Sun, L. & Chen, Z. J. (2004) Curr. Opin. Cell Biol. 16, 119–126.
29. Aguilar, R. C. & Wendland, B. (2003) Curr. Opin. Cell Biol. 15, 184–190.
30. Edwards, T. A., Wilkinson, B. D., Wharton, R. P. & Aggarwal, A. K. (2003) Genes
Dev. 17, 2508–2513.
31. Slack, F. J. & Ruvkun, G. (1998) Trends Biochem. Sci. 23, 474–475.
32. Taylor, G. S., Maehama, T. & Dixon, J. E. (2000) Proc. Natl. Acad. Sci. USA 97,
33. Taylor, G. S., Liu, Y., Baskerville, C. & Charbonneau, H. (1997) J. Biol. Chem. 272,
34. Orth, K., Xu, Z., Mudgett, M. B., Bao, Z. Q., Palmer, L. E., Bliska, J. B., Mangel,
W. F., Staskawicz, B. & Dixon, J. E. (2000) Science 290, 1594–1597.
35. Maehama, T., Taylor, G. S., Slama, J. T. & Dixon, J. E. (2000) Anal. Biochem. 279,
36. Abbott, D. W., Wilkins, A., Asara, J. M. & Cantley, L. C. (2004) Curr. Biol. 14,
37. Kotaja, N., Karvonen, U., Janne, O. A. & Palvimo, J. J. (2002) Mol. Cell. Biol. 22,
38. Leng, R. P., Lin, Y., Ma, W., Wu, H., Lemmers, B., Chung, S., Parant, J. M., Lozano,
G., Hakem, R. & Benchimol, S. (2003) Cell 112, 779–791.
Physiol. 137, 13–30.
40. Lorick, K. L., Jensen, J. P., Fang, S., Ong, A. M., Hatakeyama, S. & Weissman, A. M.
(1999) Proc. Natl. Acad. Sci. USA 96, 11364–11369.
Science 286, 309–312.
42. Borden, K. L. (2000) J. Mol. Biol. 295, 1103–1112.
43. Jackson, P. K., Eldridge, A. G., Freed, E., Furstenthal, L., Hsu, J. Y., Kaiser, B. K.
& Reimann, J. D. (2000) Trends Cell Biol. 10, 429–439.
44. Yaron, A., Hatzubai, A., Davis, M., Lavon, I., Amit, S., Manning, A. M., Andersen,
J. S., Mann, M., Mercurio, F. & Ben-Neriah, Y. (1998) Nature 396, 590–594.
45. Skowyra, D., Craig, K. L., Tyers, M., Elledge, S. J. & Harper, J. W. (1997) Cell 91,
46. Feldman, R. M., Correll, C. C., Kaplan, K. B. & Deshaies, R. J. (1997) Cell 91,
47. Zheng, N., Wang, P., Jeffrey, P. D. & Pavletich, N. P. (2000) Cell 102, 533–539.
48. Albert, T. K., Hanzawa, H., Legtenberg, Y. I., de Ruwe, M. J., van den Heuvel, F. A.,
Collart, M. A., Boelens, R. & Timmers, H. T. (2002) EMBO J. 21, 355–364.
49. Zheng, N., Schulman, B. A., Song, L., Miller, J. J., Jeffrey, P. D., Wang, P., Chu, C.,
Koepp, D. M., Elledge, S. J., Pagano, M., et al. (2002) Nature 416, 703–709.
50. Printen, J. A., Brady, M. J. & Saltiel, A. R. (1997) Science 275, 1475–1478.
51. Fong, N. M., Jensen, T. C., Shah, A. S., Parekh, N. N., Saltiel, A. R. & Brady, M. J.
(2000) J. Biol. Chem. 275, 35034–35039.
52. Brady, M. J. & Saltiel, A. R. (2001) Recent Prog. Horm. Res. 56, 157–173.
53. Liu, A. W., Delgado-Escueta, A. V., Serratosa, J. M., Alonso, M. E., Medina, M. T.,
Gee, M. N., Cordova, S., Zhao, H. Z., Spellman, J. M. & Peek, J. R. (1995) Am. J.
Hum. Genet. 57, 368–381.
54. Thomas, D. & Tyers, M. (2000) Curr. Biol. 10, R341–R343.
55. Lipford, J. R. & Deshaies, R. J. (2003) Nat. Cell Biol. 5, 845–850.
www.pnas.org?cgi?doi?10.1073?pnas.0503285102Gentry et al.