Doublecortin interacts with the ubiquitin protease DFFRX, which associates with microtubules in...

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Mol. Cell. Neurosci. 28 (2005) 153–164

Doublecortin interacts with the ubiquitin protease DFFRX,

which associates with microtubules in neuronal processes

GaJlle Friocourt,a Caroline Kappeler,a Yoann Saillour,a Fabien Fauchereau,aManuel S. Rodriguez,b

Nadia Bahi,a Marie-Claude Vinet,a Philippe Chafey,a Karine Poirier,a Shinichiro Taya,c

Stephen A. Wood,d,e Catherine Dargemont,b Fiona Francis,a,* and Jamel Chellya

aLaboratoire de Genetique et Physiopathologie des Retards Mentaux, GDPM, Institut Cochin, 75014 Paris, FrancebEquipe de transport nucleocytoplasmique, Institut Jacques Monod, CNRS UMR 7592, 75005 Paris, FrancecDepartment of Cell pharmacology, Graduate school of Medecine, Nagoya University, Nagoya 466-8550, JapandChild Health Research Institute, North Adelaide SA 5006, AustraliaeCentre for the Molecular Genetics of Development, University of Adelaide, SA 5005, Australia

Received 28 May 2004; revised 7 September 2004; accepted 13 September 2004Available online 11 November 2004

Doublecortin (DCX) is a microtubule-associated protein involved in

neuronal migration, which causes X-linked lissencephaly and subcort-

ical laminar heterotopia (SCLH)whenmutated. Here we show that DCX

interacts with the ubiquitin-specific protease Drosophila fat facets

related on X chromosome (DFFRX). This interaction was confirmed

by targeted mutagenesis, colocalization, and immunoprecipitation

studies. DFFRX is thought to deubiquitinate specific substrates includ-

ing B-catenin, preventing their degradation by the proteasome. Interest-

ingly, unlike B-catenin, no ubiquitinated forms of DCX could be

detected, and indeed we show that DCX interacts with a novel

recognition domain in DFFRX, located outside of its catalytic site. We

also show that DFFRX associates with microtubules at specific

subcellular compartments, including those enriched in DCX. These

results thus suggest that in addition to vesicular trafficking, DCX may

play a role in the regulation of cell adhesion via its interaction with

DFFRX in migrating and differentiating neurons.

D 2004 Elsevier Inc. All rights reserved.

Introduction

The complex laminar structures of the mature cerebral cortex

arise through a series of carefully regulated developmental events,

including neurogenesis, neuronal migration, and differentiation, as

well as the formation of synaptic connections. During cortico-

genesis, postmitotic neurons exit the neuroepithelium and migrate to

the cortical plate where they eventually form the mature cortical

layers. They settle in six layers sequentially formed from the deepest

1044-7431/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2004.09.005

* Corresponding author. Laboratoire de Genetique et Physiopathologie

des retards mentaux, GDPM, Institut Cochin, 24, rue du Faubourg Saint

Jacques, 75014 Paris, France. Fax: +33 1 44 41 24 21.

E-mail address: [email protected] (F. Francis).

Available online on ScienceDirect (www.sciencedirect.com).

to the most superficial (binside–outQ) so that each new wave of

neurons migrates past the previously formed ones (Hatten, 1999).

Neuronal migration is thus a key step in cortical layer organization.

Neither the molecular events that drive migrating neuronal cells to

their final destinations nor the environmental cues responsible for

the regulation of their spatial position are fully understood.

However, significant contributions to the dissection of several

pathways involved in these processes have been provided through

the genetic analysis of disorders associated with brain cortical

dysgeneses. Such disorders including type-I lissencephaly and

subcortical laminar heterotopia (SCLH), which result from muta-

tions in the LIS1 (Reiner et al., 1993) and doublecortin genes (also

called DBCN or DCX) (des Portes et al., 1998; Gleeson et al., 1998),

are characterized by a dramatic disorganization of neocortical layers

and gyral abnormalities. In addition to LIS1 and DCX, mutations in

cdk5 (Oshima et al., 1996), p35 (Chae et al., 1997), filamin 1 (Fox et

al., 1998), reelin (D’Arcangelo et al., 1995; Hong et al., 2000),

mDab1 (Howell et al., 1997; Sheldon et al., 1997;Ware et al., 1997),

VLDLR and ApoER2 (Trommsdorff et al., 1999), POMGnT1

(Michele et al., 2002; Moore et al., 2002) and POMT1 (Beltran-

Valero de Bernabe et al., 2002), and ARX (Kitamura et al., 2002)

have been found to lead to specific phenotypes associated with

disrupted corticogenesis, either in human or in mouse.

One signaling pathway is represented by Reelin, mDab1,

ApoER2, and VLDLR, which are potentially involved in the

control of the final position of neurons and function through a

linear pathway at the plasma membrane (D’Arcangelo et al.,

1999; Hiesberger et al., 1999; Trommsdorff et al., 1999).

Signaling pathways regulated by Cdk5 and its specific activator

p35 were shown to be involved in neuronal migration and several

other processes such as neurite outgrowth, vesicular trafficking,

and neurodegeneration (Smith and Tsai, 2002; Tan et al., 2003).

The Cdk5/p35 kinase has been shown to regulate N-cadherin-

mediated adhesion, which also requires h-catenin, isolated as a

G. Friocourt et al. / Mol. Cell. Neurosci. 28 (2005) 153–164154

p35-interacting protein in a two-hybrid screen (Kesavapany et al.,

2001; Kwon et al., 2000). More recently this kinase has also been

shown to phosphorylate DCX (Tanaka et al., 2004) and one of

the LIS1-interacting proteins (Niethammer et al., 2000). The

strikingly similar phenotypes of lissencephaly in humans with

mutations in LIS1 or DCX, together with findings demonstrating

that both proteins are microtubule-associated proteins (MAPs;

Francis et al., 1999; Gleeson et al., 1999; Horesh et al., 1999;

Sapir et al., 1997), suggest that LIS1 and DCX may function

through similar mechanisms. Although a recent elegant study

using an RNAi-based approach showed that DCX-deficient

neurons prematurely stop migrating to form subcortical band

heterotopias within the intermediate zone and then white matter

(Bai et al., 2003), basic molecular, biochemical, and biological

processes underlying this premature arrest of migration remain to

be elucidated.

Towards this aim, we carried out a yeast two-hybrid screen to

identify proteins interacting with DCX. Using such a screen, we

previously showed that DCX interacts with the A subunits of the

clathrin adaptor complexes AP-1 and AP-2, known to be

involved in vesicle trafficking associated with the trans-Golgi

network and the plasma membrane, respectively (Friocourt et al.,

2001). We describe here the identification and characterization of

an additional DCX-interacting protein, the ubiquitin protease

Drosophila fat facets related gene on X chromosome (DFFRX),

also called Usp9x), which is involved in cleaving ubiquitin from

protein–ubiquitin conjugates. Besides its role in protein degrada-

tion, ubiquitination has been shown to play an important function

in an increasing number of processes including endocytosis,

signaling, DNA repair, and cell cycle regulation. Monoubiquiti-

nation is in particular associated with the regulation of the

internalization of membrane proteins and their transport to the

lysosomes (Hicke, 2001). Polyubiquitination on the other hand is

necessary for protein degradation by the 26S proteasome, a multi-

subunit proteolytic complex. The deubiquitination process is

catalyzed by proteases called deubiquitinating enzymes, which

fall into four distinct families: ubiquitin C-terminal hydrolases,

ubiquitin-specific processing proteases (USPs or UBPs), out-

domain ubiquitin–aldehyde binding proteins, and Jab1/pad1/

MPN-domain-containing metalloenzymes (Kim et al., 2003).

Among these four families, UBPs represent the most widespread

deubiquitinating enzymes across evolution.

In this study, we found that DCX interacts with a novel

recognition domain in DFFRX, a member of the UBPs family. A

specific lissencephaly causing mutation in DCX leads to the loss of

this interaction. Our data show that the DFFRX mouse orthologue

associates with microtubules in specific subcellular compartments

and is thus likely to play a role in dynamic processes required

during cell migration.

Results

DCX interacts with the C-terminal part of DFFRX in yeast

As the MAP function of DCX does not fully explain the role

of this protein in neuronal migration, we searched for further

DCX-interacting proteins. Using the full-length cDNA of DCX as

bait, we screened a human fetal brain cDNA library using the

two-hybrid system (Fields and Song, 1989). Two successive

screens of this library were performed; the first one identified a

clone that contains the last 255 amino acids of human DFFRX

(DFFRX-Cter, amino acids 2292–2547), whereas the second

isolated a similar but longer C-terminal clone (the last 343 amino

acids). The specificity of the interaction with DCX was confirmed

by testing these two clones with unrelated baits (lamin C and

RAS) (Fig. 1A).

The DFFRX protein contains the typical hallmarks of the UBPs

family of deubiquitinating enzymes, that is, the presence of

cysteine and histidine boxes in a core enzymatic domain (amino

acids 1550–1948) (Jones et al., 1996). Neither the N-terminal part

of DFFRX nor the C-terminus that includes the DCX-interacting

region contain any known functional domains. DCX contains a

microtubule binding domain, composed of two repeats (amino

acids 47–135 and 174–259) (Sapir et al., 2000; Taylor et al., 2000)

and a serine/proline-rich region in its C-terminal part. To map the

domain of interaction with DFFRX in DCX, we tested several

deletion mutants with the clone DFFRX-Cter in a two-hybrid assay

and concluded that the domain of interaction is located between the

amino acids 110 and 214 that contains the proximal part of the

second microtubule binding repeat (Fig. 1B). The A1 subunit of theAP-1 complex was used as a control since we had previously

shown that A1 interacts with a YLPL motif in the C-terminus of

DCX (aa 345–348) (Friocourt et al., 2001). Different mutant clones

of DCX corresponding to missense mutations found in patients

with lissencephaly were similarly tested. Two-hybrid results

showed a loss of interaction between the clone DFFRX-Cter and

the mutant clone R192W (Fig. 1C). The position of this amino acid

and the results obtained with the DCX deletion mutants are

consistent with the defined interacting domain localized between

amino acids 110 and 214. Other mutants, also located in this region

(R186C, R196S, and T203R) still interacted with DFFRX-Cter,

suggesting that the residue R192 is particularly important for this

interaction.

Dffrx is expressed in the developing nervous system

As the pattern of expression of Dffrx, the DFFRX mouse

orthologue, has not been studied in depth in the nervous system,

we performed in situ hybridization studies using mouse embryo

sections at different stages using a 3VUTR probe of Dffrx. A probe

was chosen in the 3VUTR in order to avoid cross hybridization with

other similar UBP family members. Dffrx appears to be expressed

ubiquitously at E12.5 and E14.5 but is interestingly most strongly

expressed in the nervous system in the region of the third, fourth,

and lateral ventricles and in the developing spinal cord (Fig. 2A,

panels A and G). At E14.5, the Dffrx signal is particularly obvious

in the olfactory lobe (Fig. 2A, panels G–I) and in certain ganglia

(trigeminal ganglia, inferior glossopharyngeal ganglia, and dorsal

root ganglia; Fig. 2A, panel G). At E16.5, Dffrx expression is

clearly regionalized to the nervous system. It is strongly expressed

in the developing brain, in the mantle layer of the spinal cord, and

in certain ganglia (Fig. 2A, panels C–F). Some expression was also

detected in the testis. In the developing cortex, Dffrx is expressed

in the neuroblasts of the ventricular zone, as well as in migrating

and differentiating neurons of the intermediate zone and cortical

plate (Fig. 2A, panels J–K). In the eye, the pigment layer of the

retina and the inner neural layer of the optic cup are specifically

labeled (Fig. 2A, panels L–M). Dffrx is still strongly expressed

ubiquitously in mouse newborn brain but its expression seems to

decrease after neonatal stages and it appears generally less

abundant in most regions of the adult brain (Fig. 2B, panels A–

Fig. 1. Two-hybrid analysis. (A) Confirmation of the specificity of the interaction between DCX and DFFRX-Cter (corresponding to the last 255 residues of

DFFRX). Mating was performed between L40 transformants (indicated on left side) and AMR70 (indicated above). X-gal and histidine results are presented.

RAS/RAF: positive control. (B) Definition of DCX-interacting domain. Different constructs of DCX were tested with DFFRX-Cter and with A1 in a two-hybridassay. The identified DCX domain that interacts with DFFRX-Cter spans the residues 110–214. R1 and R2 represent the two repeats present in the microtubule

binding domain. (C) Two-hybrid results obtained with the R192W DCX mutation (identified in a patient with lissencephaly). R192W was the only DCX

mutation that showed a loss of interaction with DFFRX-Cter. Note that this mutant is still capable to interact with the A1 protein.

G. Friocourt et al. / Mol. Cell. Neurosci. 28 (2005) 153–164 155

D), although regions of the hippocampus remain intensely stained

(Fig. 2B, panels C–D). Nevertheless Western blot analysis of

mouse adult brain extracts confirmed that Dffrx is still relatively

ubiquitous (Fig. 2C). These data thus suggest that Dffrx is

important both during neuronal development and in mature

neurons.

Dffrx and DCX interact in vivo

Prior to assessing the interaction between Dffrx and Dcx in

vivo and to further support the two-hybrid results, we further

analyzed the spatial and temporal coexpression and subcellular

colocalization of Dcx and Dffrx. By Western blot analysis, we

observed that Dffrx is expressed in differentiating neurons in

primary culture, in glial and PC12 cells, but is barely detectable

in COS-7 cells (Fig. 3A). Previous studies have shown that

DCX is also expressed in young differentiating neurons, but not

in glial or PC12 cells (Francis et al., 1999; Gleeson et al., 1999;

Horesh et al., 1999). We also performed immunolabelings using

E16.5 mouse embryonic and adult brain sections. The results

obtained with antibodies directed against the catalytic domain

and against the C-terminus of Dffrx were identical and coherent

with those generated by in situ hybridization. Within neuronal

cells, Dffrx seems to be expressed in the soma as well as in the

axons, as shown in the Purkinje cells of the adult cerebellum

(Fig. 3B, panel A). In the embryonic cortex, Dffrx antibody

labels the proliferating cells of the ventricular zone, the

intermediate zone containing neurons in migration and axonal

processes, and the differentiating neurons of the cortical plate

(Fig. 3B, panels B–C). According to previous studies (Francis et

al., 1999; Gleeson et al., 1999), Dcx is present in both the

intermediate zone and the cortical plate regions of the

developing cortex. In the adult brain, Dcx is only expressed

in a limited population of neurons, which migrate from the

subventricular zone to the olfactory bulb (rostral migratory

stream), whereas Dffrx is more widely expressed. A colocaliza-

tion was observed between Dffrx and Dcx in these migrating

neurons (Fig. 3B, panels D–F). Immunolabeling using primary

cultures of cortical neurons confirmed that Dffrx was present in

both the soma and the neurites (Fig. 3C, panel A). It was

strongly expressed in growth cones and at the end of neurites,

where it partially colocalized with Dcx (Fig. 3C, panels D–I).

Double-labeling experiments performed with actin and a-tubulin

showed Dffrx expression in the neurites and the main body of

the growth cone. Growth cones are actin-rich but also contain

microtubules, and indeed Dffrx showed a colocalization with

tubulin rather than with actin, as shown here by the Dcx

labeling (Fig. 3C, panels D–I).

In order to confirm the interaction between Dffrx and Dcx in

vivo, we first performed co-immunoprecipitation experiments in

COS-7 cells transfected with FLAG-tagged DCX and GFP-tagged

DFFRX-Cter (equivalent to the clone isolated in the two-hybrid

assay). GFP-DFFRX-Cter was co-immunoprecipitated with

FLAG-DCX but not with the mutant form FLAG-R192W (Fig.

4A). These results thus confirm those obtained with the yeast two-

hybrid system. As Dffrx is an enzyme, we also looked for an

interaction between its catalytic domain and DCX. A full-length

Dffrx construct tagged with V5, or the catalytic domain of Dffrx

(amino acids 1210–2410) tagged with HA, were expressed with

either GFP-DCX or GFP alone. Immunoprecipitation with anti-

bodies directed against V5 or HA showed that DCX interacted with

full-length Dffrx but was not able to co-immunoprecipitate with the

catalytic domain of Dffrx (Fig. 4B). We then performed co-

immunoprecipitation experiments involving the endogenous pro-

teins using newborn mouse brain extracts. We selected this stage of

development because both Dffrx and Dcx remain well expressed in

Fig. 2. Expression of Dffrx during development. In situ hybridization was performed using mouse sections. (A) Dffrx is ubiquitous at E12.5 (A) and E14.5 (G)

but regionalizes to the nervous system at E16.5 (C–F). These results are likely to be specific since the sense probes show no labeling (B, D, and F). At E14.5,

Dffrx is strongly expressed in the developing olfactory bulb (H–I, I, toluidine blue coloration of the section). In the developing cortex, at E16.5, Dffrx is

expressed in the cortical plate, the intermediate zone, and the ventricular zone (J–K). In the eye of an E16.5 embryo, Dffrx is strongly expressed in the pigment

layer of the retina and in the inner nuclear layer of the optic cup (L–M, M, sense probe showing nonspecific labeling of the lens). drg: dorsal root ganglion, tg:

trigeminal ganglion, cx: cortex, ob: olfactory bulb, cp: cortical plate, iz: intermediate zone, vz: ventricular zone, le: lens, pl: pigment layer, inl: inner neural

layer. (B) Localization of Dffrx in newborn and adult mouse. Dffrx is strongly expressed throughout the newborn brain (coronal sections A–B, B, sense probe).

The absence of signal in a region of the visual cortex is probably an artefact as it was not observed in other experiments. In a coronal section of adult mouse

brain, Dffrx expression appears strongest in regions of the hippocampus (C–D, D, sense probe), although it is still expressed ubiquitously. Sagittal sections of

the adult cerebellum show a strong labeling of the Purkinje cells (E–F, F, sense probe showing nonspecific labeling of the white matter). ML: molecular layer,

GL: granular layer, P: Purkinje cells, WM: white matter. (C) Western blot analysis of protein extracts derived from different regions of the mouse adult brain.

Twenty micrograms of proteins was loaded in each well. Extracts were detected with anti-DFFRX-Cter and anti-actin (control) antibodies.


newborn brain. Dffrx was found to co-immunoprecipitate with Dcx

using anti-Dcx antibodies but was not present in anti-GST

precipitates used as a control (Fig. 4C). Dcx was not detected in

anti-Dffrx immunoprecipitates in vivo but the results with tagged

proteins are still in favor of the existence of the interaction. These

results therefore confirm that Dcx and Dffrx can exist in the same

protein complex in mouse brain in vivo and that Dcx probably

interacts only with the C-terminus of Dffrx but not with its catalytic

domain.

Dcx is not ubiquitinated and is not a substrate of Dffrx

To elucidate whether Dcx is a substrate of Dffrx and thus

could be normally regulated by ubiquitination, we first

measured the half-life of Dcx in primary cultures of rat

embryonic cortical neurons and found it to be longer than 12

h (data not shown). We next examined the effect of proteasome

inhibitors on DCX stability. PC12 cells were transfected with a

DCX expression plasmid and then treated with MG132 and

lactacystin. Under these conditions, both proteasome inhibitors

had little effect on the stability of DCX, whereas the amount of

h-catenin, a protein known to be ubiquitinated and degraded by

the proteasome (Aberle et al., 1997), strongly increased with the

presence of several slower migrating species likely resulting

from ubiquitin modifications of this protein in the presence of

MG132 and lactacystin (Fig. 5A). Similar results were obtained

using primary neuronal cultures expressing endogenous Dcx and

h-catenin (data not shown).

To further assess whether Dcx can be ubiquitinated, COS-7

cells were transfected with HA-tagged ubiquitin (HA-Ub) and

GFP-tagged DCX. GFP-p35 was used as a control since this

protein has been previously described to be ubiquitinated and

degraded by the proteasome (Patrick et al., 1998). The

expression levels of GFP-DCX were similar to those obtained

for GFP-p35 in these experiments. High molecular weight

isoforms of p35 likely corresponding to polyubiquitinated

species of this protein clearly accumulated in the presence of

MG132, whereas no modification of GFP-DCX could be

detected under the same conditions (Fig. 5B). To enrich for

further potential ubiquitinated species of p35 and DCX,

corresponding cell lysates were immunoprecipitated with anti-

HA or anti-GFP antibodies (Fig. 5C). High molecular weight

Fig. 3. Coexpression of Dffrx and Dcx during development and subcellular localization of both proteins. (A) Coexpression of Dffrx and Dcx in young

differentiating neurons in culture. Anti-N-term and anti-Dffrx antibodies were used to probe extracts from primary cultures of mouse brain neurons grown for 2,

6, 14, 21, and 30 days as well as glial cells, PC12, and COS-7 cells. (B) Immunohistochemistry performed using mouse sections. Dffrx is expressed in the soma

and in the axons of Purkinje cells in a sagittal section of an adult cerebellum (A). In the developing cortex (coronal section) of an E16.5 embryo, Dffrx is

expressed in the progenitor cells of the ventricular zone (VZ), in the intermediate zone (IZ), and in the cortical plate (CP) (B–C). In the adult brain, cells in the

rostral migratory stream that express Dcx are also stained with Dffrx (D–F), although Dffrx is more widely expressed. (C) Localization of Dffrx in

differentiating neurons derived from E18 fetal rat brain. Dffrx labels both the soma and neurites of neurons in culture, whereas Dcx more specifically labels the

extremities of neurites. Although Dffrx expression seems to extend further into the growth cone than Dcx, it is also present at the end of neurites where it

colocalizes with Dcx. The scale bar equals 25 Am.


species of p35 were immunoprecipitated with both GFP and

anti-HA antibodies confirming that they correspond to p35

ubiquitin conjugates. As expected, in these experiments, treat-

ment with MG132 increased the amount of ubiquitinated p35.

Unlike p35, we observed no higher molecular weight bands

corresponding to DCX in the anti-GFP or anti-HA precipitates

(Fig. 5C). Similarly, polyubiquitinated Dcx forms were not

detected in proteins extracts complemented with proteasome

inhibitors from E15.5 and newborn mouse brains (data not

shown). Though these results cannot totally exclude the absence

of a ubiquitinated fraction of endogenous Dcx, together they

suggest that Dcx is not a major substrate for ubiquitination and

degradation by the proteasome and consequently not a substrate

of Dffrx.

Fig. 4. Interaction between Dffrx and Dcx in vivo. (A) Co-immunoprecipitation of DFFRX-Cter with Dcx but not with the mutant R192W. FLAG-DCX or

R192W were transfected with GFP-DFFRX-Cter in COS-7 cells. After immunoprecipitation of the lysates with DCX anti-N-term or anti-DFFRX-Cter

antibodies, the blots were probed with either goat anti-DCX C18 or anti-GFP. (B) Absence of an interaction between DCX and the catalytic domain of Dffrx.

Full-length Dffrx (V5-Dffrx-FL) or its catalytic domain (HA-Dffrx-CAT) were cotransfected with either GFP-DCX or GFP alone. Lysates were

immunoprecipitated with anti-V5 or anti-HA antibodies and the blots were detected with anti-Dffrx-CAT or with anti-DCX-N-term antibodies. SAP:

supernatant after immunoprecipitation, IP: immunoprecipitation. (C) Immunoprecipitation between Dcx and Dffrx from mouse newborn brain. Extracts were

immunoprecipitated with either anti-DCX N-term, anti-GST, or anti-Dffrx-CAT antibodies and the blot was detected with anti-Dffrx-CAT.


The DCX/Dffrx complex is associated with microtubules

Immunoprecipitation experiments and colocalization of Dcx,

Dffrx, and microtubules at the distal part of neurites in differ-

entiating neurons (Fig. 3) led us to hypothesize that the function of

Dffrx may be important when it is localized in protein complexes

that interact with microtubules. The association between endoge-

nous Dffrx and microtubules was thus further investigated in

different cell types. In nondifferentiated proliferating PC12 cells,

Dffrx is concentrated in actin-rich but also microtubule-containing

structures, such as the filopodia and the midbody of dividing cells

during cytokinesis (Figs. 6A–C). In the soma, Dffrx appears to be

most concentrated in a region close to the nucleus. Double labeling

with antibodies directed against g-tubulin suggests that this region

corresponds to the centrosome or to the microtubule-organizing

center (MTOC) (Figs. 6D–F). In dividing cells, Dffrx seems to be

most concentrated at the mitotic spindle (Figs. 6G–I), which

confirms Dffrx association with the MTOC. A similar enrichment

of Dffrx in the region of the MTOC was observed in glial cells

(Figs. 6J–L) and in differentiating neurons in culture (data not

shown). Interestingly, in confluent glial cells, Dffrx showed a

different subcellular localization as it was instead concentrated at

cell–cell junctions, where it colocalized with h-catenin (Figs. 6M–

O), as previously described in MDCKII cells (Taya et al., 1999).

This observation led us to test whether Dcx and h-catenin might be

associated in the same protein complex in vivo. To this end, we

performed co-immunoprecipitation experiments from mouse

embryonic brain using anti-Dcx and anti-Dffrx antibodies and

tested for the presence of h-catenin by Western blot analysis. Fig. 7

shows that h-catenin is co-immunoprecipitated with both Dcx and

Dffrx, suggesting that these three proteins can associate in the same

protein complex in embryonic brain. The different subcellular

Fig. 5. Effect of proteasome inhibitors on DCX, h-catenin, and p35 stability. (A) Stability of transfected DCX in PC12 cells. Transfected cells were treated with

MG132 or lactacystin and the stability of DCX was compared with the stability of endogenous h-catenin. Whereas an increase in the amount of h-catenin and

of higher molecular weight products were detectable for h-catenin, very little change was observed for DCX. (B) Stability of transfected GFP-DCX in

comparison with transfected GFP-p35. COS-7 cells were cotransfected with HA-Ub and either GFP-p35 or GFP-DCX and treated with MG132 or DMSO as a

control. Detections were performed with anti-p35 and DCX anti-N-term antibodies. A p35 ubiquitin ladder appears with MG132 but no change is observed for

DCX. (C) Immunoprecipitation of ubiquitinated products. COS-7 cells were transfected as previously described, treated with MG132 or DMSO, and lysates

were immunoprecipitated with anti-GFP or anti-HA antibodies. Blots were detected with anti-DCX and anti-p35. Unlike p35, no higher molecular weight

species were observed for DCX.


localizations of Dffrx thus suggest that it is recruited to particular

regions of the cell containing dynamic components of the

cytoskeleton. Its localization in neurons in the vicinity of the

growth cone is one such example.

These data were further confirmed biochemically in polymer-

ization–depolymerization and sedimentation experiments using

tubulin enriched from brain (Francis et al., 1999). A proportion of

native Dffrx was found to cosediment with newly formed micro-

tubules in the same fractions as Dcx (lanes P1 and P2, Fig. 8).

These results together with the fact that DCX was shown to be a

microtubule-associated protein suggest that DCX could be

involved in the recruitment to and/or the regulation of Dffrx in

association with microtubules at the extremities of neuronal

processes in migrating and differentiating neurons.

Discussion

In this study, we isolated human DFFRX in a two-hybrid screen

using DCX as a bait. We showed that a missense mutation in DCX

detected in a patient with lissencephaly abolished this interaction.

The occurrence in vivo and the specificity of this interaction was

further reinforced by colocalization and co-immunoprecipitation

studies.

Several studies suggest the importance of Dffrx during

development. The Drosophila orthologue of DFFRX, the fat facets

(faf) gene is responsible for two different phenotypes when

mutated (Fischer-Vize et al., 1992; Huang and Fischer-Vize,

1996): (i) an absence of cellularization in the zygote due to a

defect in nuclear migration during oogenesis, and (ii) the presence

of ectopic photoreceptors in the compound eye, which suggests

that fat facets could positively regulate the activity of an inhibitor

of neuronal cell fate. Substrates of this ubiquitin protease involved

in these defects remain to be fully elucidated but genetic and

biochemical studies suggest that fat facets could play a role in

endocytosis by controlling the deubiquitination of liquid facets, the

Drosophila orthologue of epsin (Cadavid et al., 2000; Chen et al.,

2002). Further studies performed with mouse Dffrx, identified AF-

6, a Ras effector, and h-catenin as two additional possible

substrates (Taya et al., 1998, 1999). Up to now, DFFRX has not

been implicated in any human pathology. As DFFRX interacts with

DCX and is strongly expressed in the nervous system during

development, we screened DFFRX in 60 patients with lissence-

phaly for whom DCX and LIS1 genes were found to be normal, but

no mutations were identified. However, as Dffrx has a wider

pattern of expression than Dcx and its enzymatic activity involves

a number of different substrates, it is possible that potential

phenotypes related to its dysfunction are different and more severe

than disorders associated only with cortical dysgenesis.

The interaction between Dcx and the ubiquitin protease Dffrx

led us first to investigate the possibility that Dcx could be a

substrate for Dffrx. However, we did not observe any polyubiqui-

tinated forms of Dcx nor efficient proteasome-mediated degrada-

tion of Dcx. Both h-catenin and AF-6 were previously shown to be

Fig. 6. Dffrx expression in PC12 and glial cells. In PC12 cells (A–I), Dffrx is present in cell regions containing microtubules such as the midbody during

cytokinesis (A–C), the centrosome (D–F), and the mitotic spindle in dividing cells (G–I). In nonconfluent glial cells (J–L), Dffrx strongly labels the MTOC

(indicated by an arrow). In confluent cells, Dffrx is strongly colocalized with h-catenin at the cell–cell junctions (M–O). These data thus show a relocalization

of Dffrx depending on the cell state.


polyubiquitinated and to interact directly with a region containing

the catalytic domain of Dffrx. In contrast, Dcx interacts with a

novel recognition region in Dffrx involving its C-terminal 250

amino acids, a domain that is highly conserved among Drosophila,

mouse, and human and relatively unique to this protein. Dcx may

therefore help regulate Dffrx localization within the cell. Indeed

Dcx is a microtubule-associated protein with a specific localization

in neuronal cells (Francis et al., 1999; Gleeson et al., 1999; Horesh

et al., 1999), and Dffrx colocalizes to these regions and cosedi-

ments with microtubules. The interaction with Dcx could therefore

anchor Dffrx in specialized compartments of neuronal cells (such

as dynamic structures corresponding to distal segments of neurites)

Fig. 7. Co-immunoprecipitation of endogenous Dcx, h-catenin, and p35.

Mouse E15.5 brain extracts were immunoprecipitated with either anti-Dcx

N-term, anti-DFFRX-Cter, anti-h-catenin, or anti-p35 antibodies. h-Cateninis immunoprecipitated with Dcx, Dffrx, and in small amounts with p35.

Fig. 8. Interaction between Dffrx and microtubules. Native Dffrx is present

in microtubules fractions induced through cycles of polymerization and

depolymerization. Western blots (20 Ag protein/lane) contain total mouse

brain extracts (T), soluble (S0), and insoluble cell components (P0). The

tubulin in the S0 fraction was polymerized to form microtubules and

sedimented, resulting in soluble (S1) and pellet (P1) fractions. Microtubules

in P1 were depolymerized by incubation at 08C for 1 h and resedimented

(PV1), and the resulting soluble fraction was subjected to a further cycle of

polymerization, followed by sedimentation (S2 and P2). Like Dcx, Dffrx

cosediments with newly formed microtubules. Note that in addition to the

band corresponding to the full-length Dffrx protein, anti-Dffrx CAT

antibody detected a product with higher molecular weight than Dffrx. It

remains to be seen whether this protein corresponds to another isoform of

Dffrx or to a cross-reaction of the antibody with an as yet unidentified

protein. p35 was used as a negative control. Unlike Dffrx and DCX, it is not

present in the P2 fraction.


to regulate the activity and/or the stability of relevant proteins in

this region. A similar situation has been evoked for the MID1

microtubule-associated protein, which is likely to target phospha-

tase 2A for degradation (Trockenbacher et al., 2001). It is also

interesting to note that DCLK1C, a protein similar to Dcx, which is

expressed during development and in adult stages in astroglial and

neuronal cells (Burgess et al., 1999; Friocourt et al., 2003), also

interacts with Dffrx (data not shown). It is therefore possible that

DCLK1C could, at relevant times, aid the localization of Dffrx in

certain cells and in cellular compartments where Dcx is not

expressed although this remains to be further investigated.

Besides its potential role anchoring Dffrx to a specific local-

ization, Dcx may also influence the enzymatic activity of Dffrx.

Although it has been reported that human HAUSP is necessary and

sufficient for the in vitro deubiquitination of its specific substrate,

the p53 tumor suppressor (Li et al., 2002), the yeast Ubp3 protein

has been shown to require an additional regulatory subunit, Bre5p,

to insure its function (Cohen et al., 2003a,b). As for the interaction

of Dcx with Dffrx, Bre5p interacts with Ubp3p on a domain

distinct from the catalytic region (M. Cohen and C. Dargemont,

personal data). It is thus possible that DCX acts as a regulatory

subunit of Dffrx, aiding the deubiquitination and stabilization of

specific substrates in the vicinity of the growth cone. Indeed, the

importance of the role of the ubiquitination and protein degradation

in growth cones has been shown for the Netrin-1 signaling

pathway (Campbell and Holt, 2001).

Our co-immunoprecipitation data confirm that Dcx is present in

the same protein complexes as Dffrx and h-catenin in the

developing brain. We observed a colocalization between Dffrx

and h-catenin at cell–cell junctions in confluent glial cells and at

adherens junctions in proliferating cells of the ventricular zone

(data not shown), perhaps suggesting that Dffrx ensures the

stability of h-catenin while homotypic adhesion is required. Dcx

is not expressed in proliferating cells; nevertheless, its influence on

h-catenin stability in postmitotic neurons remains a possibility. It is

interesting to point out that despite the apparent diversity of

ubiquitinated proteins shown to be substrates of Dffrx in cultured

cellular models, all are involved directly or indirectly in cellular

processes such as cell adhesion, endocytosis, and vesicle traffick-

ing. Notably, these functions are extensively required at the

extremities of neuronal processes where Dcx is localized. AF-6,

for example, is implicated in adhesion and has a specific neuronal

isoform (Ikeda et al., 1999; Mandai et al., 1997). In addition, epsin

is involved in endocytosis and vesicle formation (Wendland,

2002). Interestingly, it has been reported that epsin binds to

microtubules in neurons and could play a role in neuritic outgrowth

(Hussain et al., 2003). In addition, a recent study shows the

presence of Dffrx at multiple points of protein trafficking in

epithelial cell lines (Murray et al., 2004). Thus, the known

substrates of Dffrx and possible functions of Faf predicted from

the phenotypes of the Drosophila mutants seem collectively to

suggest the involvement of the Dffrx/Dcx complex in the

regulation of adhesion and membrane turnover, which are essential

processes for neuronal migration and differentiation. In line with

this function are the previously reported data indicating that DCX

interacts with the A1 and A2 subunits of the clathrin adaptor

complexes implicated in vesicle trafficking (Friocourt et al., 2001)

and the phosphorylated form of neurofascin, a protein implicated in

neuronal adhesion (Kizhatil et al., 2002). Determining the relation-

ships between these different partners of DCX will further

elucidate its role in neuronal migration and differentiation.

Experimental methods

Yeast cultures and two-hybrid analysis

The human fetal brain cDNA library cloned in pACT2 was

purchased from CLONTECH laboratories (MATCHMAKER) and

transformed into the L40 yeast strain. A first screen was performed

by transforming the full-length DCX cDNA cloned in pBTM116

into the L40 strain containing the library plasmids (Friocourt et al.,

2001), and the second one was performed by mating between the

L40DGal4 strain transformed with VJL12-DCX and the Y187

strain transformed with the library.

The mutant forms of DCX (S47R, R59L, Y125H, W146C,

R186C, R196S, and T203R) were generated using the Quik

Change Mutagenesis kit (Stratagene). The constructs correspond-


ing to the catalytic domain of Dffrx (aa 1210–2410) and to Dffrx

full-length cDNA have been previously described (Murray et al.,

2004; Taya et al., 1998).

Antibodies

We used affinity-purified antibodies directed against the N-

terminal part (anti-N-term; Francis et al., 1999) and the C-terminal

part of DCX (Santa Cruz, C18). A polyclonal antibody directed

against the C-terminal part of DFFRX (anti-DFFRX-Cter, encom-

passing amino acids 2292-2547 according to GenBank X98296)

was generated and affinity purified. Towards this aim, a two-hybrid

clone containing DFFRX-Cter was used to generate a GST fusion

protein in bacteria. The protein obtained was purified by

chromatography using glutathione–sepharose and used to generate

rabbit polyclonal antisera. In this region, the amino acid sequence

is 98% identical between human and mouse. This antibody detects

by immunoblotting a band at the expected size. The specificity of

this band was confirmed by comparison with polyclonal antibodies

directed against the catalytic domain of mouse Dffrx (anti-Dffrx-

CAT) and against the first 20 N-terminal amino acids, gifts from Pr

K. Kaibuchi and S.A. Wood, respectively. The following anti-

bodies were also used: polyclonal anti-p35 (Santa Cruz) and

monoclonal anti-GFP (Roche), anti-actin (Chemicon), anti-HA

(Babco), a-tubulin, and g-tubulin (Sigma); anti-h-catenin anti-

bodies (Transduction Laboratories); and rat anti-HA antibodies

(Roche).

In situ hybridization

Mouse Dffrx 3’UTR sequence (nucleotides 9052–9983,

U67874) was cloned in pBluescript, and sense and antisense

probes were generated by in vitro transcription. Mouse embryos

and adult brains were fixed in 2–4% paraformaldehyde, cryopro-

tected with 30% sucrose in phosphate buffer, frozen with

isopentane, and cut into approximately 20 Am thick sections.

Slides were incubated with a35S-labelled RNA probes in a 50%

formamide solution at 608C. Washes were performed in succes-

sively stringent SSC solutions, with a final wash at 0.1� SSC at

608C. Slides were then dipped in diluted Kodak NTB2 emulsion

and exposed for 24–48 h. Emulsions were developed and sections

counterstained with toluidine blue, mounted in Eukitt, and

examined under light microscopy with an SMZ1000 stereoscopic

microscope (Nikon) and an Axiophot microscope (Zeiss).

Immunohistochemistry and cytochemistry

Mouse embryos and adult brains were frozen with isopentane

and sectioned (approximately 10 Am thick). Sections were fixed at

48C for 20 min in 4% paraformaldehyde solution. Primary

neuronal cultures were prepared from E18 rat brains as previously

described (Berwald-Netter et al., 1981). Neurons grown on

coverslips were fixed for 7 min at �208C in methanol. Immuno-

fluorescence labeling was performed as previously described

(Friocourt et al., 2001).

Primary glial cultures were prepared according to Nowak et al.

(1987) from newborn OF1 mouse cerebral hemispheres. Glial cells

were cultured in Dulbecco’s modified Eagle’s medium (DMEM,

Invitrogen) supplemented with 4.5 mg/ml glucose, 10% fetal

bovine serum (Invitrogen), and 100 units/ml of penicillin and

streptomycin. They were used as secondary cultures containing

more than 95% of GFAP-positive astrocytes. After three washes in

phosphate-buffered saline solution (PBS), the cells were prehybri-

dized for 1 h with TBST containing 2% goat serum or BSA, and

then incubated overnight at 48C with the primary antibody.

Co-immunoprecipitation

COS-7 cells were cultured in DMEM (Invitrogen) supple-

mented with 10% fetal bovine serum. Cells were transfected using

the FUGENE6 transfection reagent (Roche Molecular Biochem-

icals), lysed after 24 h in IP buffer (50 mM Tris–HCl pH 8, 150

mM NaCl, 1% NP40, and protease inhibitors), and centrifuged at

50,000 � g for 30 min at 48C. After a preclearing with protein A-

agarose (Sigma), aliquots of the resulting supernatant were

incubated overnight at 48C with the antibodies of interest. Protein

A-agarose was added and samples were incubated 2 h at 48C.Immunoprecipitates were washed with IP buffer and proteins were

separated on SDS-polyacrylamide gels.

Mouse E15.5 embryonic or newborn brains were crushed in

liquid nitrogen and resuspended in 10 volumes of IP buffer.

Immunoprecipitations were performed as described above.

Ubiquitination studies

PC12 cells were grown in DMEM supplemented with 5% fetal

bovine serum and 10% horse serum and were transfected with

GFP-DCX using GenePorter (gene Therapy System) according to

the manufacturer’s instructions. Twenty hours after transfection,

the proteasome inhibitors lactacystin (Calbiochem) and MG132

(Affiniti) were added at the final concentrations of 12.5 AM for 2 h

and 20 AM for 5 h 30 min, respectively. Cells were harvested and

resuspended in sample buffer.

For immunoprecipitation experiments, COS-7 cells were

transfected with the different constructs. Twenty hours after

transfection, cells were treated with 20 AM of MG132 for 5 h and

lysed in IP buffer complemented with iodoacetic acid and N-

ethylmaleimide (Sigma, final concentrations 10 and 5 mM,

respectively). Immunoprecipitations were performed as described

above.

Sedimentation of microtubules

The temperature-dependent polymerization–depolymerization

experiment was performed as previously described (Francis et al.,

1999). A brain fraction enriched in tubulin was prepared from E20

mouse embryos by homogenization in MEM buffer (50 mM MES

pH 6.8, 2 mM EGTA, 2 mM MgCl2) with protease inhibitors and

ultracentrifugation (150,000 � g at 48C for 1 h). Soluble tubulin

(S0) was incubated in the presence of 1 mM GTP and 25%

glycerol for 40 min at 378C to form microtubules that were

sedimented (150,000 � g) for 60 min at 308C. The resulting pellet

(P1) was depolymerized for 60 min at 08C and gently homogen-

ized. Sedimentation of the insoluble (PV1) from soluble components

was performed. This soluble fraction was polymerized and

sedimented as before giving rise to P2 and S2 fractions.

Acknowledgments

The authors are grateful to K. Kaibuchi, D. Boucher, M.

Bornens, and A. Benmerah for kind gifts of reagents and helpful


discussions, Pierre Billuart, Sebastien L’Hoste, Philippe Couvert,

Evelyne Souil, Jean-Marie Genevard, and Emmanuelle Tisserand

for their technical contribution to the work, and Odette Godard

for her logistic support. This work was supported in part by

grants from INSERM, CNRS, the Fondation Bettencourt-Schuler,

the Fondation pour la Recherche Medicale, the European

Commission (No. QLG3-CT-2002-01810), the Human Frontier

Science Program (ref. RG0283/1999-B), the Ministere de la

Recherche (ACI-1A066G), and the Federation pour la Recherche

sur le Cerveau. G.F. was supported by a PhD fellowship from the

Fondation pour la Recherche Medicale and by the Fondation

Bettencourt-Schuler.

References

Aberle, H., Bauer, A., Stappert, J., Kispert, A., Kemler, R., 1997.

h-catenin is a target for the ubiquitin–proteasome pathway. EMBO

J. 16, 3797–3804.

Bai, J., Ramos, R.L., Ackman, J.B., Thomas, A.M., Lee, R.V., LoTurco,

J.J., 2003. RNAi reveals doublecortin is required for radial migration in

rat neocortex. Nat. Neurosci. 6, 1277–1283.

Beltran-Valero de Bernabe, D., Currier, S., Steinbrecher, A., Celli, J., van

Beusekom, E., van der Zwaag, B., Kayserili, H., Merlini, L., Chitayat,

D., Dobyns, W.B., Cormand, B., Lehesjoki, A.E., Cruces, J., Voit, T.,

Walsh, C.A., van Bokhoven, H., Brunner, H.G., 2002. Mutations in the

O-mannosyltransferase gene POMT1 give rise to the severe neuronal

migration disorder Walker–Warburg syndrome. Am. J. Hum. Genet. 71,

1033–1043.

Berwald-Netter, Y., Martin-Moutot, N., Koulakoff, A., Couraud, F., 1981.

Na+-channel-associated scorpion toxin receptor sites as probes for

neuronal evolution in vivo and in vitro. Proc. Natl. Acad. Sci. U. S. A.

78, 1245–1249.

Burgess, H.A., Martinez, S., Reiner, O., 1999. KIAA0369, doublecortin-

like kinase, is expressed during brain development. J. Neurosci. Res.

58, 567–575.

Cadavid, A.L., Ginzel, A., Fischer, J.A., 2000. The function of the

Drosophila fat facets deubiquitinating enzyme in limiting photoreceptor

cell number is intimately associated with endocytosis. Development

127, 1727–1736.

Campbell, D.S., Holt, C.E., 2001. Chemotropic responses of retinal growth

cones mediated by rapid local protein synthesis and degradation.

Neuron 32, 1013–1026.

Chae, T., Kwon, Y.T., Bronson, R., Dikkes, P., Li, E., Tsai, L.H., 1997.

Mice lacking p35, a neuronal specific activator of cdk5, display

cortical lamination defects, seizures, and adult lethality. Neuron 18,

29–42.

Chen, X., Zhang, B., Fischer, J.A., 2002. A specific protein substrate for a

deubiquitinating enzyme: liquid facets is the substrate of Fat facets.

Genes Dev. 16, 289–294.

Cohen, M., Stutz, F., Belgareh, N., Haguenauer-Tsapis, R., Dargemont, C.,

2003a. Ubp3 requires a cofactor, Bre5, to specifically de-ubiquitinate

the COPII protein, Sec23. Nat. Cell Biol. 5, 661–667.

Cohen, M., Stutz, F., Dargemont, C., 2003b. De-ubiquitination: a new

player in Golgi to ER retrograde transport. J. Biol. Chem. 278,

51989–51992.

D’Arcangelo, G., Miao, G.G., Chen, S.C., Soares, H.D., Morgan, J.I.,

Curran, T., 1995. A protein related to extracellular matrix proteins

deleted in the mouse mutant reeler. Nature 374, 719–723.

D’Arcangelo, G., Homayouni, R., Keshvara, L., Rice, D.S., Sheldon, M.,

Curran, T., 1999. Reelin is a ligand for lipoprotein receptors. Neuron

24, 471–479.

des Portes, V., Pinard, J.M., Billuart, P., Vinet, M.C., Koulakoff, A., Carrie,

A., Gelot, A., Dupuis, E., Motte, J., Berwald-Netter, Y., Catala, M.,

Kahn, A., Beldjord, C., Chelly, J., 1998. A novel CNS gene required for

neuronal migration and involved in X-linked subcortical laminar

heterotopia and lissencephaly syndrome. Cell 92, 51–61.

Fields, S., Song, O., 1989. A novel genetic system to detect protein–protein

interactions. Nature 340, 245–246.

Fischer-Vize, J.A., Rubin, G.M., Lehmann, R., 1992. The fat facets gene is

required for Drosophila eye and embryo development. Development

116, 985–1000.

Fox, J.W., Lamperti, E.D., Eksioglu, Y.Z., Hong, S.E., Feng, Y., Graham,

D.A., Scheffer, I.E., Dobyns, W.B., Hirsch, B.A., Radtke, R.A.,

Berkovic, S.F., Huttenlocher, P.R., Walsh, C.A., 1998. Mutations in

filamin 1 prevent migration of cerebral cortical neurons in human

periventricular heterotopia. Neuron 21, 1315–1325.

Francis, F., Koulakoff, A., Boucher, D., Chafey, P., Schaar, B., Vinet, M.C.,

Friocourt, G., McDonnell, N., Reiner, O., Kahn, A., McConnell, S.K.,

Berwald-Netter, Y., Denoulet, P., Chelly, J., 1999. Doublecortin is a

developmentally regulated, microtubule-associated protein expressed in

migrating and differentiating neurons. Neuron 23, 247–256.

Friocourt, G., Chafey, P., Billuart, P., Koulakoff, A., Vinet, M.C., Schaar,

B., McConnell, S.K., Francis, F., Chelly, J., 2001. Doublecortin

interacts with A subunits of clathrin adaptor complexes in the

developing nervous system. Mol. Cell. Neurosci. 18, 307–319.

Friocourt, G., Koulakoff, A., Chafey, P., Boucher, D., Fauchereau, F.,

Chelly, J., Francis, F., 2003. Doublecortin functions at the extremities of

growing neuronal processes. Cereb. Cortex 13, 620–626.

Gleeson, J.G., Allen, K.M., Fox, J.W., Lamperti, E.D., Berkovic, S.,

Scheffer, I., Cooper, E.C., Dobyns, W.B., Minnerath, S.R., Ross, M.E.,

Walsh, C.A., 1998. Doublecortin, a brain-specific gene mutated in

human X-linked lissencephaly and double cortex syndrome, encodes a

putative signaling protein. Cell 92, 63–72.

Gleeson, J.G., Lin, P.T., Flanagan, L.A., Walsh, C.A., 1999. Doublecortin is

a microtubule-associated protein and is expressed widely by migrating

neurons. Neuron 23, 257–271.

Hatten, M.E., 1999. Central nervous system neuronal migration. Annu.

Rev. Neurosci. 22, 511–539.

Hicke, L., 2001. Protein regulation by monoubiquitination. Nat. Rev., Mol.

Cell Biol. 2, 195–201.

Hiesberger, T., Trommsdorff, M., Howell, B.W., Goffinet, A., Mumby,

M.C., Cooper, J.A., Herz, J., 1999. Direct binding of Reelin to VLDL

receptor and ApoE receptor 2 induces tyrosine phosphorylation of

disabled-1 and modulates tau phosphorylation. Neuron 24, 481–489.

Hong, S.E., Shugart, Y.Y., Huang, D.T., Shahwan, S.A., Grant, P.E.,

Hourihane, J.O., Martin, N.D., Walsh, C.A., 2000. Autosomal recessive

lissencephaly with cerebellar hypoplasia is associated with human

RELN mutations. Nat. Genet. 26, 93–96.

Horesh, D., Sapir, T., Francis, F., Wolf, S.G., Caspi, M., Elbaum, M.,

Chelly, J., Reiner, O., 1999. Doublecortin, a stabilizer of microtubules.

Hum. Mol. Genet. 8, 1599–1610.

Howell, B.W., Hawkes, R., Soriano, P., Cooper, J.A., 1997. Neuronal

position in the developing brain is regulated by mouse disabled-1.

Nature 389, 733–737.

Huang, Y., Fischer-Vize, J.A., 1996. Undifferentiated cells in the develop-

ing Drosophila eye influence facet assembly and require the Fat facets

ubiquitin-specific protease. Development 122, 3207–3216.

Hussain, N.K., Yamabhai, M., Bhakar, A.L., Metzler, M., Ferguson,

S.S., Hayden, M.R., McPherson, P.S., Kay, B.K., 2003. A role for

ENTH/ANTH domains in tubulin binding. J. Biol. Chem. 278,

28823–28830.

Ikeda, W., Nakanishi, H., Miyoshi, J., Mandai, K., Ishizaki, H., Tanaka, M.,

Togawa, A., Takahishi, K., Nishioka, H., Yoshida, H., Mizoguchi, A.,

Nishikawa, S., Takai, Y., 1999. Afadin: a key molecule essential for

structural organization of cell–cell junctions of polarized epithelia

during embryogenesis. J. Cell Biol. 146, 1117–1132.

Jones, M.H., Furlong, R.A., Burkin, H., Chalmers, I.J., Brown, G.M.,

Khwaja, O., Affara, N.A., 1996. The Drosophila developmental gene

fat facets has a human homologue in Xp11.4 which escapes X-

inactivation and has related sequences on Yq11.2. Hum. Mol. Genet. 5,

1695–1701.


Kesavapany, S., Lau, K.F., McLoughlin, D.M., Brownlees, J., Ackerley, S.,

Leigh, P.N., Shaw, C.E., Miller, C.C.J., 2001. p35/cdk5 binds and

phosphorylates h-catenin and regulates h-catenin/presenilin-1 interac-

tion. Eur. J. Neurosci. 13, 241–247.

Kim, J.H., Park, K.C., Chung, S.S., Bang, O., Chung, C.H., 2003.

Deubiquitinating enzymes as cellular regulators. J. Biochem. 134, 9–18.

Kitamura, K., Yanazawa, M., Sugiyama, N., Miura, H., Iizuka-Kogo, A.,

Kusaka, M., Omichi, K., Suzuki, R., Kato-Fukui, Y., Kamiirisa, K.,

Matsuo, M., Kamijo, S.I., Kasahara, M., Yoshioka, H., Ogata, T.,

Fukuda, T., Kondo, I., Kato, M., Dobyns, W.B., Yokoyama, M.,

Morohashi, K.I, 2002. Mutation of ARX causes abnormal development

of forebrain and testes in mice and X-linked lissencephaly with

abnormal genitalia in humans. Nat. Genet. 32, 359–369.

Kizhatil, K., Wu, Y., Sen, A., Bennett, V., 2002. A new activity of

Doublecortin in recognition of the phospho-FIGQY tyrosine in the

cytoplasmic domain of Neurofascin. J. Neurosci. 22, 7948–7958.

Kwon, Y.T., Gupta, A., Zhou, Y., Nikolic, M., Tsai, L.H., 2000. Regulation

of N-cadherin-mediated adhesion by the p35-cdk5 kinase. Curr. Biol.

10, 363–372.

Li, M., Chen, D., Shiloh, A., Luo, J., Nikolaev, A.Y., Qin, J., Gu, W., 2002.

Deubiquitination of p53 by HAUSP is an important pathway for p53

stabilization. Nature 416, 648–653.

Mandai, K., Nakanishi, H., Satoh, A., Obaishi, H., Wada, M., Nishioka, H.,

Itoh, M., Mizoguchi, A., Aoki, T., Fujimoto, T., Matsuda, Y., Tsukita,

S., Takai, Y., 1997. Afadin: a novel actin filament-binding protein with

one PDZ domain localized at cadherin-based cell-to-cell adherens

junction. J. Cell Biol. 139, 517–528.

Michele, D.E., Barresi, R., Kanagawa, M., Saito, F., Cohn, R.D., Satz, J.S.,

Dollar, J., Nishino, I., Kelley, R.I., Somer, H., Straub, V., Mathews,

K.D., Moore, S.A., Campbell, K.P., 2002. Post-translational disruption

of dystroglycan-ligand interactions in congenital muscular dystrophies.

Nature 418, 417–422.

Moore, S.A., Saito, F., Chen, J., Michele, D.E., Henry, M.D., Messing,

A., Cohn, R.D., Ross-Barta, S.E., Westra, S., Williamson, R.A.,

Hoshi, T., Campbell, K.P., 2002. Deletion of brain dystroglycan

recapitulates aspects of congenital muscular dystrophy. Nature 418,

422–425.

Murray, R.Z., Jolly, L.A., Wood, S.A., 2004. The FAM deubiquitilating

enzyme localizes to multiple points of protein trafficking in epithelia,

where it associates with E-cadherin and h-catenin. Mol. Biol. Cell 15,

1591–1599.

Niethammer, M., Smith, D.S., Ayala, R., Peng, J., Ko, J., Lee, M.S.,

Morabito, M., Tsai, L.H., 2000. NUDEL is a novel Cdk5

substrate that associates with LIS1 and cytoplasmic dynein. Neuron

28, 697–711.

Nowak, L., Ascher, P., Berwald-Netter, Y., 1987. Ionic channels in mouse

astrocytes in culture. J. Neurosci. 7, 101–109.

Oshima, T., Ward, J.M., Huh, C.G., Longenecker, G., Veeranna, Pant, H.C.,

Brady, R.O., Martin, L.J., Kulkarni, A.B., 1996. Targeted disruption of

the cyclin-dependent kinase 5 gene results in abnormal corticogenesis,

neuronal pathology and perinatal death. Proc. Natl. Acad. Sci. U. S. A.

93, 11173–11178.

Patrick, G.N., Zhou, P., Kwon, Y.T., Howley, P.M., Tsai, L.H., 1998. p35,

the neuronal-specific activator of cyclin-dependent kinase 5 (cdk5) is

degraded by the ubiquitin-proteasome pathway. J. Biol. Chem. 273,

24057–24064.

Reiner, O., Carrozzo, R., Shen, Y., Wehnert, M., Faustinella, F., Dobyns,

W.B., Caskey, C.T., Ledbetter, D.H., 1993. Isolation of a Miller–Dieker

lissencephaly gene containing G protein beta-subunit-like repeats.

Nature 364, 717–721.

Sapir, T., Elbaum, M., Reiner, O., 1997. Reduction of microtubule

catastrophe events by LIS1, platelet-activating factor acetylhydrolase

subunit. EMBO J. 16, 6977–6984.

Sapir, T., Horesh, D., Caspi, M., Atlas, R., Burgess, H.A., Wolf, S.G.,

Francis, F., Chelly, J., Elbaum, M., Pietrokovski, S., Reiner, O., 2000.

Doublecortin mutations cluster in evolutionarily conserved functional

domains. Hum. Mol. Genet. 9, 703–712.

Sheldon, M., Rice, D.S., D’Arcangelo, G., Yoneshima, H., Nakajima, K.,

Mikoshiba, K., Howell, B.W., Cooper, J.A., Goldowitz, D., Curran, T.,

1997. Scrambler and yotari disrupt the disabled gene and produce a

reeler-like phenotype in mice. Nature 389, 730–733.

Smith, D.S., Tsai, L.H., 2002. Cdk5 behind the wheel: a role in trafficking

and transport? Trends Cell Biol. 12, 28–36.

Tan, T.C., Valova, V.A., Malladi, C.S., Graham, M.E., Berven, L.A., Jupp,

O.J., Hansra, G., McClure, S.J., Sarcevic, B., Boadle, R.A., Larsen,

M.R., Cousin, M.A., Robinson, P.J., 2003. Cdk5 is essential for

synaptic vesicle endocytosis. Nat. Cell Biol. 5, 701–710.

Tanaka, T., Serneo, F.F., Tseng, H.C., Kulkarni, A.B., Tsai, L.H., Gleeson,

J.G., 2004. Cdk5 phosphorylation of doublecortin ser297 regulates its

effect on neuronal migration. Neuron 41, 215–227.

Taya, S., Yamamoto, T., Kano, K., Kawano, Y., Iwamatsu, A., Tsuchiya, T.,

Tanaka, K., Kanai-Azuma, M., Wood, S.A., Mattick, J.S., Kaibuchi, K.,

1998. The Ras target AF-6 is a substrate of the fam deubiquitinating

enzyme. J. Cell Biol. 142, 1053–1062.

Taya, S., Yamamoto, T., Kanai-Azuma, M., Wood, S.A., Kaibuchi, K.,

1999. The deubiquitinating enzyme Fam interacts with and stabilizes h-catenin. Genes Cells 4, 757–767.

Taylor, K.R., Holzer, A.K., Bazan, J.F., Walsh, C.A., Gleeson, J.G., 2000.

Patient mutations in doublecortin define a repeated tubulin-binding

domain. J. Biol. Chem. 275, 34442–34450.

Trockenbacher, A., Suckow, V., Foerster, J., Winter, J., Krauss, S., Ropers,

H., Schneider, R., Schweiger, S., 2001. MID1, mutated in Opitz

syndrome encodes an ubiquitin ligase that targets phosphatase 2A for

degradation. Nat. Genet. 29, 287–294.

Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W.,

Nimpf, J., Hammer, R.E., Richardson, J.A., Herz, J., 1999. Reeler/

Disabled-like disruption of neuronal migration in knockout mice

lacking the VLDL receptor and ApoE receptor 2. Cell 97, 689–701.

Ware, M.L., Fox, J.W., Gonzalez, J.L., Davis, N.M., Lambert de Rouvroit,

C., Russo, C.J., Chua, S.C.J., Goffinet, A.M., Walsh, C.A., 1997.

Aberrant splicing of a mouse disabled homolog, mdab1, in the

scrambler mouse. Neuron 19, 239–249.

Wendland, B., 2002. Epsins: adaptors in endocytosis? Nat. Rev., Mol. Cell

Biol. 3, 971–977.

Doublecortin interacts with the ubiquitin protease DFFRX, which associates with microtubules in...

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