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ALS2/Alsin Regulates Rac-PAK Signaling and
Neurite Outgrowth*□S
Received for publication, June 7, 2005, and in revised form, July 25, 2005 Published, JBC Papers in Press, July 26, 2005, DOI 10.1074/jbc.M506216200
Elizabeth L. Tudor‡§1, Michael S. Perkinton‡§, Anja Schmidt¶, Steven Ackerley‡§, Janet Brownlees‡§,
Nicholas J. O. Jacobsen‡§, Helen L. Byers, Malcolm Ward, Alan Hall¶, P. Nigel Leigh§, Christopher E. Shaw§,
Declan M. McLoughlin**, and Christopher C. J. Miller‡§2
From the Departments of ‡Neuroscience and §Neurology and **Section of Old Age Psychiatry, Institute of Psychiatry, Kings College,
London SE5 8AF, United Kingdom, ¶Medical Research Council Laboratory for Molecular Cell Biology, University College, London
WC1E 6BT, United Kingdom, and Proteome Sciences plc, Institute of Psychiatry, London SE5 8AF, United Kingdom
Rac and its downstream effectors p21-activated kinase (PAK)
family kinases regulate actin dynamics within growth cones to control neurite outgrowth during development. The activity of Rac is
stimulated by guanine nucleotide exchange factors (GEFs) that promote GDP release and GTP binding. ALS2/Alsin is a recently
described GEF that contains a central domain that is predicted to
regulate the activities ofRac and/orRho andCdc42 activities.Mutations in ALS2 cause some recessive familial forms of amyotrophic
lateral sclerosis (ALS) but the function of ALS2 is poorly understood. Here we demonstrate that ALS2 is present within growth
cones of neurons, in which it co-localizes with Rac. Furthermore,
ALS2 stimulates Rac but not Rho or Cdc42 activities, and this
induces a corresponding increase in PAK1 activity. Finally, we demonstrate that ALS2 promotes neurite outgrowth. Defects in these
functionsmay therefore contribute tomotor neurondemise inALS.
Some forms of amyotrophic lateral sclerosis (ALS)3 are familial and
are passed through generations in autosomal dominant, recessive, or
X-linked fashions (1, 2). Mutations in the gene encoding copper/zinc
superoxide dismutase-1 (SOD1) cause someof these familial cases (3, 4),
and recently, mutations in the ALS2/Alsin gene have been shown to
cause some rare juvenile forms of ALS (5, 6). Mutations in ALS2 have
also been linked to juvenile primary lateral sclerosis and infantile-onset
ascending hereditary spastic paraplegia (7–9).
The structure of ALS2 predicts that it functions as a guanine nucleotide exchange factor (GEF). GEFs regulate the activity of members of
the Ras superfamily of GTPases. These GTPases cycle between inactive
(GDP-bound) and active (GTP-bound) conformational states, and
GEFs stimulate GTP-binding so as to promote activation of the GTPase
(10, 11). ALS2 contains three putative GEF domains: an amino-terminal
domain that displays homology to the Ran GEF RCC1; a central region
containing Dbl and pleckstrin homology (DH/PH) domains that are
found in GEFs for Rho, Rac, and Cdc42; and a carboxyl-terminal vacuolar protein-sorting 9 (VPS9) domain that is found in GEFs for Rab5 (5,
6). Indeed, there is now evidence that ALS2 functions as a GEF, including one for Rab5 that is via its VPS9 domain (12–15).
The mechanisms by which mutant ALS2 induces disease are poorly
understood. One possibility is that there are links between ALS2 and
mutant SOD1 and that there are common pathways of toxicity (15, 16).
However, the recessive nature and types of mutations in affected families strongly suggest that a loss of ALS2 function is the primary cause of
disease. Indeed, at least some of the mutants generate unstable forms of
the protein (17). A proper understanding of the molecular mechanisms
by which mutant ALS2 induces motor neuron disease thus requires
insight into ALS2 function. Here we demonstrate that ALS2 stimulates
Rac1-p21-activated kinase (PAK) signaling and is involved in neurite
Plasmids andMutagenesis—Acarboxyl-terminalmyc-tagged human
ALS2 cDNA was generated by PCR. Briefly, the 5-end of ALS2 was
amplified by PCR from a human brain cDNA library and ligated to a
5-truncated partial ALS2 cDNA (clone KIAA1563) so as to create a
full-length clone. A carboxyl-terminal myc tag was then added by PCR,
and the tagged full-length cDNA was then cloned into pCIneo (Promega) as a NotI fragment. A mutant ALS2 clone (ALS2DH) in which
the DH/PH GEF domain was disrupted was created by the deletion of
sequences encoding the DH domain (residues 747–826). Sequences
were deleted using an ExSite mutagenesis kit (Stratagene) with primers
CTTGCTGAATCGGCTAGC-3. ALS2 phosphorylation sites were
altered using a QuikChange Multi site-directed mutagenesis kit (Stratagene) and primers 5-GCCAGCACTGCTCTCGCCCCCTCCACTGAAACC-3 (S277A), 5-GATTGTTGTCACAAGTTGCCCCCAG-
domain (amino acids 681–1010) of ALS2 was generated by PCR and
cloned as a BamHI-EcoRI fragment into pRK5myc. RhoA, Rac1,
L61Rac1 (constitutively active Rac1), N17Rac1 (dominant-negative
Rac1), Cdc42, and N39Rab5 (dominant-negative Rab5) were all
expressed using pRK5myc variants. pCMV6myc-PAK1 clone was
obtained from Sashi Kesavapany (National Institutes of Health,
Bethesda, MD). Vector pCIneoCAT expressing the Escherichia coli
chloramphenicol acetyl transferase gene (18) was used as a control for
* This work was supported by grants from the Medical Research Council and Wellcome
Trust (to C. C. J. M.). The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□S The on-line version of this article (available at contains supplemental Figs. S1–S4.
1 Supported by a Jim Tew Memorial Studentship and the UK Motor Neurone Disease
2 To whom correspondence should be addressed: Dept. of Neuroscience, P. O. Box P037,
The Institute of Psychiatry, De Crespigny Park, Denmark Hill, London SE5 8AF, United
Kingdom. Tel.: 44-2078480393; Fax: 44-2077080017; E-mail: chris.miller@iop.
3 The abbreviations used are: ALS, amyotrophic lateral sclerosis; GEF, guanine nucleotide
exchange factor; DIV, day(s) in vitro; GFP, green fluorescent protein; CAT, chloramphenicol acetyltransferase; PAK, p21-activated kinase; RBD, Rho-binding domain;
PBD, p21-binding domain; SOD1, superoxide dismutase-1; DH, Dbl homology; PH,
pleckstrin homology; GST, glutathione S-transferase; CHO, Chinese hamster ovary;
TBS, Tris-HCl-buffered saline; VPS9, vacuolar protein-sorting 9; Cdk, cyclin-dependent
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 41, pp. 34735–34740, October 14, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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comparisons of the effects of ALS2 expression on GTPase and PAK1
activities and neurite outgrowth. Plasmid pEGFPC.1 was obtained from
Antibodies—Sequences encoding residues 452–668 of ALS2 were
TACTACAGGAGAGAAG-3 and cloned into pGEX5X1 as an EcoRI
fragment. Glutathione S-transferase (GST)-ALS2-(452–668) was
expressed in E. coli BL21-CodonPlus(DE3)RIL, purified essentially
according to the manufacturer’s instructions (Amersham Biosciences),
and used to immunize rabbits. The ALS2 antibody was affinity-purified
against antigen prior to use. Antibodies to PAK1 and to myc tags (antibody 9B11) were obtained fromNew England Biolabs; Rac, Rho(A,B,C),
and Cdc42 antibodies were from Upstate; Rab5 antibody was from BD
Biosciences; -tubulin antibody (DM1A) was from Sigma; AlexaFluor
568-phalloidin was from Molecular Probes.
Cell Culture and Transfection—CHO cells were grown in HAM’s
F-12 medium containing 10% (v/v) fetal bovine serum supplemented
with 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). Cells were transfected using Lipofectamine
(Invitrogen) according to the manufacturer’s instructions. Primary cortical and hippocampal neurons were obtained from embryonic day 18
rat embryos and cultured on glass coverslips coated with poly-D-lysine
in 12-well plates (Falcon) in neurobasal medium and B27 supplement
(Invitrogen) containing 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine. Neurons were transfected using Lipofectamine 2000 (Invitrogen) as described previously (19).
SDS-PAGE and Immunoblotting—Samples were processed for SDSPAGE by the addition of SDS-PAGE sample buffer and heating immediately in a boiling water bath. Proteins were separated on 10% (w/v)
acrylamide gels and transferred to Protran nitrocellulose membranes
(Schleicher & Schuell) using a Bio-Rad TransBlot system. Following
blocking and probing with primary antibodies, the blots were washed
and incubated with horseradish peroxidase-conjugated anti-mouse or
anti-rabbit Ig (Amersham Biosciences) and developed using an
enhanced chemiluminescence system (AmershamBiosciences) according to the manufacturer’s instructions.
GTPase and PAK1 Assays—Cellular Rho, Rac, and Cdc42 activities
were assayed using commercially available kits essentially according to
the manufacturer’s instructions (Upstate Biotechnology). Briefly, CHO
cells were co-transfected with RhoA, Rac1, or Cdc42 in combination
with ALS2 plasmids or vector encoding CAT (pCIneoCAT) as a negative control. Cells were harvested into ice-cold lysis buffer (composed of
25mMHEPES (pH 7.5), 150mMNaCl, 1%Nonidet P-40, 10 mMMgCl2,
1mMEDTA, 10% glycerol, 1mMNaF, 1mM sodium orthovanadate, and
complete protease inhibitor mixture (Roche Applied Science)) 24 h
posttransfection and following 16 h of serum starvation. Active (GTPbound) Rho, Rac, and Cdc42 were captured on GST-bait beads. Active
Rho was captured using GST-rhotekin Rho-binding domain (GSTRBD) and active Rac andCdc42 captured usingGST-PAK1p21-binding
domain (GST-PBD). Captured Rho, Rac, and Cdc42 were detected on
immunoblots, and the relative amounts were quantified by pixel densitometry using a Bio-Rad GS710 imaging densitometer and Quantity 1
software as described previously (20).
PAK1 activities in transfected CHO cells were assayed essentially as
described previously by us for other kinases (21, 22). Briefly, cells were
co-transfected with PAK1, Rac, and ALS2, ALS2DH, or vector encoding CAT as a negative control. For a positive control, cells were cotransfected with PAK1 and a constitutively active Rac (L61Rac1). Cells
wereharvested into ice-cold lysis buffer (composedof 20mMHEPES (pH7.4),
orthovanadate, 50 mM -glycerophosphate, and complete protease inhibitor
mixture (Roche Applied Science)), and following preclearing of the samples,
PAK1 was isolated by immunoprecipitation. In vitro kinase assays were performed at 30 °C in 25mMHEPES, 20mMMgCl2, 20mM-glycerophosphate,
20mM p-nitrophenylphosphate, 0.1mM sodiumorthovanadate, 2mMdithiothreitol,0.26MBq[-32P]ATP,20MATP,and5gofmyelinbasicproteinas
substrate ina finalvolumeof30l.Thereactionswere terminatedafter20min
(which pilot studies had demonstrated was within the linear range of PAK1
activity) by the addition of SDS-PAGE sample buffer and heating in a boiling
water bath, and the samples were separated by SDS-PAGE. The gels were
stainedwithCoomassieBlue, and a radioactive signalwas detectedusingFujix
autoradiography. A proportion of the reaction was also probed for immunoprecipitated PAK1 on immunoblots so as to demonstrate equal amounts of
kinase in the different transfections.
Immunofluorescence Studies and Neurite Length Measurements—
Neurons grown on coverslips were fixed in 4% (w/v) paraformaldehyde
in Tris-HCl-buffered saline (TBS) for 20 min, permeabilized in 0.1%
(w/v) Triton X-100 in TBS for 10 min, blocked with 5% (v/v) goat
serum/0.2% (w/v) Tween 20 in TBS for 1 h, and then probed with primary antibodies diluted in blocking solution. Primary antibodies were
then detected using goat anti-mouse or goat anti-rabbit Ig coupled to
AlexaFluor 350 or AlexaFluor 546 (Molecular Probes), and the samples
were mounted in Vectashield (Vector Laboratories).
For analyses of the effects of ALS2 on neurite outgrowth, 2 DIV cortical neurons were co-transfected with plasmid pEGFPC.1 (Clontech)
expressing enhanced green fluorescent protein (GFP) plus experimental
or control plasmids.GFPwas used to determine cell shape because it has
been shown to distribute uniformly throughout neurons and has been
used as a marker for neuronal cell shape in numerous studies (e.g. Refs.
23 and 24). Experimental and control plasmids included ALS2,
ALS2DH, dominant-negative N17Rac and N39Rab5, and vector
expressing pCIneoCAT,whichwas used to balance transfections so that
all cells received the same numbers and amounts of plasmid. All of the
GFP-expressing cells were immunostained for co-transfected protein to
FIGURE 1. ALS2 stimulates Rac activity. Rho, Rac, and Cdc42 activation assays were
conducted in CHO cells transfected with Rho, Rac, or Cdc42,  ALS2 or control vector
pCIneoCAT (CAT). A, representative assays for the three GTPases. Active Rho was pulled
down from the lysates using GST-RBD, and active Rac and Cdc42 were pulled down with
GST-PBD. The bound Rho, Rac, and Cdc42 were then detected by immunoblotting
(Active). The amounts of Rho, Rac, and Cdc42 transfected into the cells were also
detected by immunoblotting so as to demonstrate equal transfection efficiencies (Total).
GST alone did not bind any GTPase in the transfected cells. B, disruption of the ALS2 Rac
GEF domain (in ALS2DH) abrogates the stimulatory effect on Rac activity. C, histogram
of fold increases in GTPase activity that were obtained from four separate experiments
for each GTPase. Error bars are S.E. One-way analysis of variance tests showed that ALS2
significantly increased Rac activity by 3.3-fold (p  0.001) but not Rho or Cdc42 activities.
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confirm expression. Only healthy cells as judged by morphology
(including nuclear staining with Hoechst 33258 (Sigma) to confirm that
nuclei had a nonapoptotic appearance) were analyzed. Cells were analyzed 24 h later; the analysis included counting the numbers and measuring the lengths of axons and dendrites. However, only the longest
neurite in each cell was used in the comparisons, which is similar to the
methods used in numerous other studies on the effects of signaling
cascades on neurite outgrowth (e.g.Refs. 25 and 26). Cells were analyzed
without knowledge of the transfected plasmids.
Conventional images were captured using a Zeiss Axioscop microscope and charge-coupled device camera (Princeton Instruments), and
confocal images were captured using a Zeiss LSM 510 META confocal
microscope. Images for neurite outgrowth experiments were analyzed
using MetaMorph image analysis software. Neurite lengths were determined as the distance from the edge of the cell body to the growth cone tip.
Mass Spectrometric Sequencing of ALS2—ALS2 was isolated by
immunoprecipitation and sequenced to identify phosphorylation sites,
essentially as described by us for other proteins (18, 27). Briefly, ALS2
was immunoprecipitated from transfected CHO cells by use of the myc
tag and then isolated by excision of bands after SDS-PAGE. Bands were
reduced, alkylated, and digested with trypsin, chymotrypsin, or Asp-N
(Roche Applied Science), and peptides were extracted with two wash
cycles of 50 mM NH4HCO3 and acetonitrile and then lyophilized and
resuspended in 20 l of 50 mM NH4HCO3.
Peptide digests were analyzed by on-line liquid chromatography tandem mass spectrometry. Peptides were ionized by electrospray ionization using a Z-spray source fitted to a QTof-micro mass spectrometer
(MicromassUK). The instrumentwas set to run in automated switching
mode, selecting precursor ions based on their intensity and charge state,
for sequencing by collision-induced fragmentation. The tandem mass
spectrometry analyses were conducted using collision energy profiles
thatwere chosen based on themass/charge (m/z) and the charge state of
the peptide and optimized for phosphorylated peptides.
The mass spectral data were processed into peak lists containing the
m/z value of each precursor ion and the corresponding fragment ion
m/z values and intensities. Data were searched against a custom-built
data base containing the full-length sequence of ALS2 using theMascot
searching algorithm (Matrix Science). Peptides and phosphopeptides of
ALS2 were identified as described previously (18, 27).
Statistical Analyses—Statistical significance for GTPase and PAK1
activities and for neurite outgrowth was determined using one-way
analysis of variance followed by Tukey post-hoc tests for pairwise comparison. Differences were considered significant at p  0.05.
ALS2 Stimulates Rac1-PAK1 Signaling—ALS2 contains a central
DH/PH domain that shows homology to GEFs that regulate Rho, Rac,
and Cdc42 GTPases. To determine whether ALS2 activates these
GTPases, we utilized in vivo pull-down assays to monitor the activities
of RhoA, Rac1, and Cdc42 GTPases in transfected CHO cells. Active
(GTP-bound) Rho binds to rhotekin, whereas active Rac and Cdc42
both bind PAK1. GST-RBD and GST-PBD ”baits“ can thus be used to
isolate GTP-bound RhoA, Rac1, and Cdc42 from experimentally
manipulated cells; the amounts of these GTPases detected on immunoblots correlates with their activities (28, 29).
Co-transfection of cells with ALS2 induced a significant (3.3-fold)
increase in the amount of Rac1 but notCdc42 pulled downbyGST-PBD
bait. Although a small increase in the amount of Rho pulled down by
GST-RBD bait was also observed in ALS2-co-transfected cells, this was
not statistically significant (Fig. 1A). To confirm that the stimulatory
effect on Rac activity was indeed mediated by the DH/PH domain, we
created an ALS2 mutant (Fig. 1B, ALS2DH) in which the majority of
the catalytic DH domain (residues 747–826) was deleted. This mutant
did not activate Rac (Fig. 1B). However, the isolated DH/PH GEF
domain (residues 681–1010) similarly did not activate Rac1 in these
assays, which suggests other regions of ALS2 may be necessary for control of its Rac GEF function (supplemental data, Fig. S1). Thus, ALS2
stimulates Rac1 activity, and this stimulation requires the full-length
ALS2 holoprotein. Recently, others have also shown that ALS2 stimulates Rac activity (13, 15).
Immediate downstream targets for Rac include members of the PAK
family of serine/threonine kinases (30). PAK1 is amajor neuronal member
of the PAK family. We therefore inquired whether ALS2 also stimulated
PAK1 activity. In vitro PAK1 assays were performed from CHO cells cotransfected with PAK1  ALS2. These experiments revealed that ALS2
stimulatedPAK1activity but that this stimulationwas lost in cells co-transfectedwith the nonfunctionalmutantALS2DH,which indicates that this
activity is dependent on a functional DH/PH (Rac GEF) domain (Fig. 2).
Thus, ALS2 functions as a GEF to regulate Rac1-PAK1 signaling.
ALS2 Is Present in Neuronal Growth Cones and Co-localizes with
Rac—Rho, Rac, and Cdc42 GTPases play a major role in organizing the
cytoskeleton and in particular the actin cytoskeleton (11, 30). In developing neurons, Rho, Rac, and Cdc42 are present in growth cones of
axons and dendrites, in which they regulate actin dynamics to control
neurite outgrowth. In some paradigms, Rac and Cdc42 promote neurite
outgrowth, whereas Rho inhibits it (30–34). PAK1 is also present within
the growth cone where it too can function in neurite outgrowth,
although its precise role may depend upon the stage and type of neuron
(30). We therefore studied developmental expression and also the subcellular distribution of ALS2 in rat cortical and hippocampal neurons.
Immunoblots of mouse brains aged from embryonic day 15 (Fig. 3A,
FIGURE 2. ALS2 stimulates PAK1 activity. PAK1 in vitro kinase assays were performed
with CHO cells co-transfected with PAK1, Rac1, and ALS2, ALS2DH, or vector pCIneoCAT (CAT). For a positive control, cells were co-transfected with a constitutively active
Rac1 (L61Rac).  and  refer to the absence or presence of PAK1 immunoprecipitating
antibody in the reactions; reaction mix (RM) contains no immunoprecipitation sample.
The top panel shows the Coomassie-stained gel with myelin basic protein substrate
(MBP), and the middle panel shows the corresponding autoradiograph of the samples.
Also shown are levels of PAK1 (bottom panel) in the samples. The assays were repeated an
additional three times, and one-way analysis of variance tests revealed that the stimulatory effect of ALS2 and L61Rac1 on PAK1 activity was significant (p  0.01).
FIGURE 3. Expression of ALS2 in brain and cultured rat cortical neurons. A, an immunoblot to demonstrate developmental expression of ALS2 in mouse brain from embryonic day 15 (E15) to postnatal (P) 1 year. B, a similar immunoblot of rat cortical neurons
cultured for 2–21 DIV as indicated. The samples were also probed for tubulin to demonstrate equal protein loadings.
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E15) to 1 year revealed the presence of similar levels of ALS2 that
migrated as a single major species of 180 kDa. This 180-kDa species
co-migratedwithALS2 in transfectedCHOcells and corresponds to the
predictedmolecularmass of ALS2.Others have also reported that ALS2
expression does not change markedly in the rodent brain after embryonic day 10 (35). Similar immunoblots of rat cortical neurons also
revealed the presence of ALS2; however, there was a small but consistent decrease in ALS2 levels in cultures that were 11 days old (Fig. 3B).
Application of the ALS2 antibody to 2 DIV rat hippocampal and
cortical neurons produced prominent staining within cell bodies; labeling of punctate structures within neurites was also detected (Fig. 4).
These results are similar to those described by others in 7 DIV rat
hippocampal neurons (13). However, in the 2–3 DIV neurons, we also
detected labeling within growth cones of both axons and dendrites (Fig.
4A); Rac and actin are known to be enriched within growth cones. We
also studied the subcellular distributions of transfected ALS2 and
ALS2DH by use of the myc tags, and these too localized to cell bodies,
axons, and dendrites, and also within growth cones, in ways that were
not noticeably different from that of endogenous ALS2 (Fig. 4B). Transfected ALS2 therefore appears to localize to the appropriate cellular
compartments in which endogenous ALS2 resides.
Growth cones are made up of two domains: the central domain and
the peripheral domain. The central domain contains microtubules,
whereas the peripheral domain is actin-rich, containing themostmotile
structures, the lamellipodia and filopodia. Co-staining for ALS2 with
tubulin or actin revealed that ALS2 was present throughout the whole
growth cone (Fig. 4, C–E). We also performed double labeling for ALS2
andRac1, and this revealed a close overlap in the distributions of the two
proteins in growth cones (Fig. 4C). These results complement the biochemical studies that demonstrate that ALS2 is a RacGEF, and together
they suggest that ALS2 may function in growth cone motility and neurite outgrowth.
ALS2 Stimulates Neurite Outgrowth—A number of GEFs that stimulate the activities of Rac, Rho, and Cdc42 GTPases have been shown to
FIGURE 4. ALS2 is present in the growth cone
and co-localizes with Rac. A, endogenous ALS2
and F-actin in 2 DIV rat hippocampal neurons; note
the presence of ALS2 in growth cones (arrows). B,
localization of transfected ALS2 and ALS2DH in
cortical neurons; again both are present in cell
bodies but also in the growth cone (arrows). C–E,
images of ALS2 and Rac (C), F-actin (D), and tubulin
(E) in growth cones as indicated. Images in A, B, D,
and E are confocal Z projections; C shows a confocal slice. Scale bars, 10 m (A and B) and 5 m (C–E).
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regulate neurite outgrowth (23, 24, 26, 36, 37). We therefore examined
the effect of overexpression of ALS2 on the development of axons and
dendrites in rat cortical neurons. To do so, we transfected 2 DIV neurons with ALS2 or control vectors and analyzed neurite outgrowth in
the cells 24 h later.
The numbers of neurites (neurite tip number per cell) were not
affected by overexpression of ALS2 or ALS2DH (supplemental data,
Fig. S2). However, ALS2 significantly increased (by 1.5-fold) the length
of the longest neurite (Fig. 5). This level of stimulation in neurite outgrowth is similar to that observed by other Rac GEFs (23). By contrast,
overexpression of ALS2DH had no effect on neurite outgrowth (Fig.
5). To determine, in addition, that the ALS2-mediated stimulation of
neurite outgrowth involves Rac signaling, we compared neurite outgrowth in cells co-transfected with ALS2 and either a dominant-negative Rac (N17Rac) or a dominant-negative Rab5 (N39Rab5). ALS2 stimulates both Rac activity via its central DH/PH domain and Rab5 activity
via its VPS9 domain (Refs. 12–15 and see above). This approach, involving dominant-negative GTPases to dissect out pathways by which other
GEFs stimulate neurite outgrowth, has been utilized successfully in
many other studies (e.g. Refs. 23 and 26). Co-transfection of ALS2 with
N17Rac but not N39Rab5 abrogated the stimulatory effect of ALS2 on
neurite outgrowth (Fig. 5). Thus, ALS2 promotes neurite outgrowth in
rat cortical neurons, and this involves its Rac but not its Rab5 GEF
Expression of N17Rac alone had no effect on neurite outgrowth,
which is consistent with a number of other reports (23, 38). However,
Rac has also been shown to both promote and inhibit neurite outgrowth, and it is likely that such conflicting results are due to the different types of neurons, ages, and culture conditions used for experimentation (e.g. Refs. 39–42). Nevertheless, our findings that N17Rac blocks
the effect of ALS2 but has no effect on neurite outgrowth alone is similar
to that seen in studies with other Rac GEFs (23, 26).
ALS2 Is a Phosphoprotein—To begin to understand the upstream
mechanisms that regulate ALS2 activity, we sequenced the protein to
identify phosphorylation sites. A number ofGEFs are known to be phosphorylated, and this can regulate their activities; this includes roles in
neurite outgrowth (e.g. Refs. 43 and 44). ALS2 is a particularly low
abundance protein (17), and so to obtain sufficient protein for sequencing, we isolated it from transfected CHO cells. Using a combination of
trypsin, chymotrypsin, and Asp-N protease digestion, we obtained 81%
sequence coverage (supplemental data, Fig. S3). Serines 277, 492, 1335,
and 1464 and threonine 510 were all unambiguously identified as phosphorylation sites; a number of other phosphopeptides were also
detected, although the responsible residues could not be identified. The
identified residues all precede a proline making them candidates for
phosphorylation by proline-directed kinases such as those of the mitogen-activated protein kinase superfamily, the cyclin-dependent kinases
(Cdks) and glycogen synthase kinase-3/. To inquire whether phosphorylation of these sites influenced ALS2 regulation of Rac activity, we
constructedmutants in which serine/threonine residues were altered to
alanine to preclude phosphorylation, but thesemutants had no discernible effect on ALS2 Rac activity, PAK1 activity, or neurite outgrowth
(supplemental data, Fig. S4).
Rho, Rac, and Cdc42 are key regulators in the development of axons
anddendrites. They achieve this by transducing upstream signaling cues
to regulate actin dynamics within the growth cone (11, 30–34). The
activities of Rho, Rac, and Cdc42 are regulated in a variety of fashions
including activation by GEFs that stimulate GTP binding and inhibition
by GTPase activating proteins that promote GTP release (10). A number of GEFs have now been shown to be involved in the development of
axons and dendrites (23, 24, 26, 36, 37).
ALS2 is a recently described protein that contains three potential
GEF domains (5, 6). The carboxyl-terminal, VPS9 homology domain
has been shown to act as a GEF for Rab5; Rab5 is an essential regulator
of endocytosis and endosome biogenesis (12, 13). The central region of
ALS2 contains Dbl homology and pleckstrin homology (DH/PH)
domains and thus resembles GEFs for Rho, Rac, and Cdc42. Immediate
downstream effectors of Rac include the PAK family kinases, and Rac1PAK1 signaling is known to modulate actin dynamics so as to regulate
the growth of both axons and dendrites (30, 32). We show here that
ALS2 is present within growth cones of both axons and dendrites and
that it acts as a GEF for Rac1 to stimulate PAK1 activity and neurite
outgrowth. The DH/PH GEF domain is essential for this signaling
because its disruption abrogates the effect on both Rac1 and PAK1
activities and on the ability of ALS2 to promote neurite outgrowth.
We also demonstrate that ALS2 is a phosphoprotein and report the
identification of five cellular phosphorylation sites. All of these are Ser/
Thr-Pro motifs, which makes them targets for proline-directed kinases
such as members of the stress-activated protein kinase family (c-Jun
amino-terminal kinases and p38) and Cdks. Interestingly, aberrant activation of both p38 and cdk5 (a neuronal Cdk) are seen in ALS and
mutant SOD1 transgenicmodels of ALS, and this activationmay be part
of mutant SOD1 toxicity (45–51). However, mutation of these sites did
not noticeably alter the effect of ALS2 on Rac activity. Whether phosphorylation regulates other ALS2 GEF (Rab5 or Ran) activities will be
addressed in future studies and when the full complement of ALS2
phosphorylation sites have been identified.
Nine disease-causing mutations in ALS2 have been described in nine
different autosomal recessive kindreds. All of the affected individuals
FIGURE 5. ALS2 promotes neurite outgrowth in cultured rat cortical neurons. 2 DIV
rat cortical neurons were transfected with GFP  control or experimental plasmids, and
the length of the longest neurite was measured using GFP as a marker. Transfections
were balanced with CAT plasmid so that all of the cells received the same number and
amount of plasmids. A, histogram of mean neurite length for each transfection condition
as indicated. Data were obtained from 40 –50 cells per transfection, and the experiments
were repeated at least three times. ALS2 but not ALS2DH stimulates neurite outgrowth
by 1.5-fold (p  0.001), and this effect is lost upon co-transfection with N17Rac but not
N39Rab5. B, representative images of cells in the different transfections: a, GFPCAT; b,
GFPALS2; c, GFPALS2DH; d, GFPALS2N17Rac; e, GFPALS2N39Rab5; f,
GFPN17Rac; g, GFPN39Rab5. Scale bar, 10 m.
ALS2 Stimulates Neurite Outgrowth
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are homozygous for the mutation and develop a slowly progressive,
ascending uppermotor neuron disorder that presents with a lower limb
spasticity and can have onset in infancy, childhood, or adolescence. All
of themutations result in premature translational termination and truncation of the full-length native protein (5–9). The neuropathology has
not been described, but in a clinically similar genetic disorder, hereditary spastic paraplegia due to mutations in SPG4, upper motor neurons
projecting into the corticospinal tract develop a dying-back axonopathy.
The recessive nature of ALS2 and truncationmutations suggest that the
disorder is caused by a loss of normal ALS2 function, but the function of
ALS2 is unclear and the precise mechanisms by which this leads to
clinically selective motor neuron degeneration are unknown.
One possibility is that the loss of ALS2 Rab5 GEF function perturbs
membrane trafficking so as to induce disease. Indeed, disruptions to
membrane trafficking and the Golgi are seen in mutant SOD1 transgenic mice models of ALS, and recently mutations in the vesicle-trafficking protein VAPB have been shown to cause late onset spinal muscular atrophy and ALS (52, 53). There may even be mechanisms linking
mutant SOD1 and ALS2 forms of ALS (15, 16).
Another possibility, and one that is supported by the findings
reported here, is that the loss of ALS2 Rac1 GEF function compromises
proper development of motor neurons making them more susceptible
to later toxic insults. Indeed, upper motor neurons are the largest in the
central nervous system with the longest axons, and so any defect in
axonal growth induced by the loss of ALS2 function is likely to be most
severe in these cells. However, none of the above hypotheses are mutually exclusive. Whatever the mechanisms by which mutations in ALS2
induce disease, a proper understanding of ALS2 function is likely to
assist in unraveling the aberrant molecular processes by which motor
neurons die in ALS.
Acknowledgments—We thank the Kazusa DNA Research Institute (Japan) for
the gift of partial ALS2 cDNA, Alison Stevenson and Boris Rogelj for tissue
samples, and Gerald Finnerty for advice on confocal imaging.
1. Figlewicz, D. A., and Orrell, R. W. (2003) Amyotroph. Lateral Scler. Other Motor
Neuron Disord. 4, 225–231
2. Bruijn, L. I., Miller, T. M., and Cleveland, D. W. (2004) Annu. Rev. Neurosci. 27,
3. Deng, H. X., Hentati, A., Tainer, J. A., Iqbal, Z., Cayabyab, A., Hung, W. Y., Getzoff,
E. D., Hu, P., Herzfeldt, B., Roos, R. P., Warner, C., Deng, G., Soriano, E., Smyth, C.,
Parge, H. E., Ahmed, A., Roses, A. D., Hallewell, R. A., Pericakvance, M. A., and
Siddique, T. (1993) Science 261, 1047–1051
4. Rosen, D. R., Siddique, T., Patterson, D., Figlewicz, D. A., Sapp, P., Hentati, A.,
Donaldson, D., Goto, J., O’Regan, J. P., Deng, H. X., and Brown, R. (1993)Nature 362,
5. Hadano, S., Hand, C. K., Osuga, H., Yanagisawa, Y., Otomo, A., Devon, R. S., Miyamoto, N., Showguchi-Miyata, J., Okada, Y., Singaraja, R., Figlewicz, D. A., Kwiatkowski, T., Hosler, B. A., Sagie, T., Skaug, J., Nasir, J., Brown, R. H., Jr., Scherer, S. W.,
Rouleau, G. A., Hayden, M. R., and Ikeda, J. E. (2001) Nat. Genet. 29, 166–173
6. Yang, Y., Hentati, A., Deng, H. X., Dabbagh, O., Sasaki, T., Hirano, M., Hung, W. Y.,
Ouahchi, K., Yan, J. H., Azim, A. C., Cole, N., Gascon, G., Yagmour, A., Ben-Hamida,
M., Pericak-Vance, M., Hentati, F., and Siddique, T. (2001) Nat. Genet. 29, 160–165
7. Gros-Louis, F., Meijer, I. A., Hand, C. K., Dube, M. P., MacGregor, D. L., Seni, M. H.,
Devon, R. S., Hayden, M. R., Andermann, F., Andermann, E., and Rouleau, G. A.
(2003) Ann. Neurol. 53, 144–145
8. Eymard-Pierre, E., Lesca, G., Dollet, S., Santorelli, F. M., di Capua, M., Bertini, E., and
Boespflug-Tanguy, O. (2002) Am. J. Hum. Genet. 71, 518–527
9. Devon, R. S., Helm, J. R., Rouleau, G. A., Leitner, Y., Lerman-Sagie, T., Lev, D., and
Hayden, M. R. (2003) Clin. Genet. 64, 210–215
10. Schmidt, A., and Hall, A. (2002) Genes Dev. 16, 1587–1609
11. Etienne-Manneville, S., and Hall, A. (2002) Nature 420, 629–635
12. Otomo, A., Hadano, S., Okada, T., Mizumura, H., Kunita, R., Nishijima, H., Showguchi-Miyata, J., Yanagisawa, Y., Kohiki, E., Suga, E., Yasuda, M., Osuga, H., Nishimoto,
T., Narumiya, S., and Ikeda, J. E. (2003) Hum. Mol. Genet. 12, 1671–1687
13. Topp, J. D., Gray, N. W., Gerard, R. D., and Horazdovsky, B. F. (2004) J. Biol. Chem.
14. Kunita, R., Otomo, A., Mizumura, H., Suzuki, K., Showguchi-Miyata, J., Yanagisawa,
Y., Hadano, S., and Ikeda, J. E. (2004) J. Biol. Chem. 279, 38625–38635
15. Kanekura, K., Hashimoto, Y., Kita, Y., Sasabe, J., Aiso, S., Nishimoto, I., andMatsuoka,
M. (2005) J. Biol. Chem. 280, 4532–4543
16. Kanekura, K., Hashimoto, Y., Niikura, T., Aiso, S., Matsuoka, M., and Nishimoto, I.
(2004) J. Biol. Chem.
17. Yamanaka, K., Vande Velde, C., Eymard-Pierre, E., Bertini, E., Boespflug-Tanguy, O.,
and Cleveland, D. W. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 16041–16046
18. Perkinton, M. S., Standen, C. L., Lau, K. F., Kesavapany, S., Byers, H. L., Ward, M.,
McLoughlin, D. M., and Miller, C. C. (2004) J. Biol. Chem. 279, 22084–22091
19. Brownlees, J., Ackerley, S., Grierson, A. J., Jacobsen, N. J., Shea, K., Anderton, B. H.,
Leigh, P. N., Shaw, C. E., and Miller, C. C. (2002) Hum. Mol. Genet. 11, 2837–2844
20. Lee, J. H., Lau, K. F., Perkinton,M. S., Standen,C. L., Rogelj, B., Falinska, A.,McLoughlin, D. M., and Miller, C. C. (2004) J. Biol. Chem. 279, 49099–49104
21. Guidato, S., McLoughlin, D., Grierson, A. J., and Miller, C. C. J. (1998) J. Neurochem.
70, 492–500
22. Brownlees, J., Yates, A., Bajaj, N. P., Davis, D., Anderton, B. H., Leigh, P. N., Shaw,
C. E., and Miller, C. C. J. (2000) J. Cell Sci. 113, 401–407
23. Bryan, B., Kumar, V., Stafford, L. J., Cai, Y., Wu, G., and Liu, M. (2004) J. Biol. Chem.
279, 45824–45832
24. May, V., Schiller, M. R., Eipper, B. A., and Mains, R. E. (2002) J. Neurosci. 22,
25. Nikolic,M., Dudek,H., Kwon, Y. T., Ramos, Y. F.M., andTsai, L. H. (1996)GenesDev.
10, 816–825
26. Penzes, P., Johnson, R. C., Kambampati, V., Mains, R. E., and Eipper, B. A. (2001)
J. Neurosci. 21, 8426–8434
27. Standen, C. L., Perkinton, M. S., Byers, H. L., Kesavapany, S., Lau, K. F., Ward, M.,
McLoughlin, D., and Miller, C. C. (2003)Mol. Cell. Neurosci. 24, 851–857
28. Benard, V., Bohl, B. P., and Bokoch, G. M. (1999) J. Biol. Chem. 274, 13198–131204
29. Ren, X. D., Kiosses, W. B., and Schwartz, M. A. (1999) EMBO J. 18, 578–585
30. Nikolic, M. (2002) Int. J. Biochem. Cell Biol. 34, 731–745
31. Dickson, B. J. (2001) Curr. Opin. Neurobiol. 11, 103–110
32. Lundquist, E. A. (2003) Curr. Opin. Neurobiol. 13, 384–390
33. Threadgill, R., Bobb, K., and Ghosh, A. (1997) Neuron 19, 625–634
34. Li, Z., Van Aelst, L., and Cline, H. T. (2000) Nat. Neurosci. 3, 217–225
35. Devon, R. S., Schwab, C., Topp, J. D., Orban, P. C., Yang, Y. Z., Pape, T. D., Helm, J. R.,
Davidson, T. L., Rogers, D. A., Gros-Louis, F., Rouleau, G., Horazdovsky, B. F., Leavitt,
B. R., and Hayden, M. R. (2005) Neurobiol. Dis. 18, 243–257
36. Estrach, S., Schmidt, S., Diriong, S., Penna, A., Blangy, A., Fort, P., and Debant, A.
(2002) Curr. Biol. 12, 307–312
37. Leeuwen, F. N., Kain, H. E., Kammen, R. A., Michiels, F., Kranenburg, O. W., and
Collard, J. G. (1997) J. Cell Biol. 139, 797–807
38. Arakawa, Y., Bito, H., Furuyashiki, T., Tsuji, T., Takemoto-Kimura, S., Kimura, K.,
Nozaki, K., Hashimoto, N., and Narumiya, S. (2003) J. Cell Biol. 161, 381–391
39. Luo, L., Liao, Y. J., Jan, L. Y., and Jan, Y. N. (1994) Genes Dev. 8, 1787–1802
40. Ruchhoeft, M. L., Ohnuma, S., McNeill, L., Holt, C. E., and Harris, W. A. (1999)
J. Neurosci. 19, 8454–8463
41. Jin, Z., and Strittmatter, S. M. (1997) J. Neurosci. 17, 6256–6263
42. Kuhn, T. B., Brown, M. D., Wilcox, C. L., Raper, J. A., and Bamburg, J. R. (1999)
J. Neurosci. 19, 1965–1975
43. Sahin,M., Greer, P. L., Lin,M. Z., Poucher, H., Eberhart, J., Schmidt, S.,Wright, T.M.,
Shamah, S.M.,O’Connell, S., Cowan, C.W.,Hu, L., Goldberg, J. L., Debant, A., Corfas,
G., Krull, C. E., and Greenberg, M. E. (2005) Neuron 46, 191–204
44. Tybulewicz, V. (2005) Curr. Opin. Immunol. 17, 267–274
45. Ackerley, S., Grierson, A. J., Banner, S., Perkinton, M. S., Brownlees, J., Byers, H. L.,
Ward, M., Thornhill, P., Hussain, K., Waby, J. S., Anderton, B. H., Cooper, J. D.,
Dingwall, C., Leigh, P. N., Shaw, C. E., and Miller, C. C. J. (2004)Mol. Cell. Neurosci.
26, 354–364
46. Nguyen, M. D., Lariviere, R. C., and Julien, J. P. (2001) Neuron 30, 135–147
47. Tortarolo,M., Veglianese, P., Calvaresi, N., Botturi, A., Rossi, C., Giorgini, A.,Migheli,
A., and Bendotti, C. (2003)Mol. Cell. Neurosci. 23, 180–192
48. Hu, J. H., Chernoff, K., Pelech, S., and Krieger, C. (2003) J. Neurochem. 85, 422–431
49. Hu, J. H., Zhang, H.,Wagey, R., Krieger, C., and Pelech, S. L. (2003) J. Neurochem. 85,
50. Raoul, C., Estevez, A., Nishimune, H., Cleveland, D., deLapeyriere, O., Henderson, C.,
Haase, G., and Pettmann, B. (2002) Neuron 35, 1067–1083
51. Zhu, S., Stavrovskaya, I. G., Drozda, M., Kim, B. Y., Ona, V., Li, M., Sarang, S., Liu,
A. S., Hartley, D. M., Wu du, C., Gullans, S., Ferrante, R. J., Przedborski, S., Kristal,
B. S., and Friedlander, R. M. (2002) Nature 417, 74–78
52. Mourelatos, Z., Gonatas,N. K., Stieber, A., Gurney,M. E., andDalCanto,M.C. (1996)
Proc. Natl. Acad. Sci. U. S. A. 93, 5472–5477
53. Nishimura, A. L., Mitne-Neto, M., Silva, H. C., Richieri-Costa, A., Middleton, S.,
Cascio, D., Kok, F., Oliveira, J. R., Gillingwater, T., Webb, J., Skehel, P., and Zatz, M.
(2004) Am. J. Hum. Genet. 75, 822–831
ALS2 Stimulates Neurite Outgrowth
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