University of Würzburg, Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Versbacherstr. 5, 97078 Würzburg, Germany
* Author for correspondence (e-mail: raabe{at}biozentrum.uni-wuerzburg.de)
Accepted 31 October 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Drosophila, Eye, Adherens junctions, Mbt/Cdc42
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All PAK proteins share a C-terminal kinase domain and a N-terminal binding
domain for proteins of the Rho family of small GTPases (p21-binding domain,
PBD). The group I PAKs show some additional structural features that are
missing in group II PAKs. Most importantly, the PBD of group I PAKs is
C-terminally flanked by the kinase inhibitory domain (KID), which negatively
regulates kinase activity through interaction with the kinase domain. Binding
of GTP-bound forms of Cdc42 or Rac releases this intramolecular association,
resulting in autophosphorylation and full activation of the kinase
(Buchwald et al., 2001;
Chong et al., 2001
;
Lei et al., 2000
). Group I
PAKs also possess several proline-rich sequences that bind to SH3
domain-containing proteins. Interaction with the SH2/SH3 domain adaptor
proteins Nck and the corresponding Drosophila homologue Dock provides
a link to cell-surface receptors (Galisteo
et al., 1996
; Hing et al.,
1999
; Lu et al.,
1997
; Lu and Mayer,
1999
; Zhao et al.,
2000
). SH3 domain-mediated binding to Cool/PIX proteins can
positively or negatively regulate PAK kinase activity
(Bagrodia et al., 1999
;
Bagrodia et al., 1998
;
Manser et al., 1998
).
Two major functions of PAK proteins have emerged: regulation of signalling
cascades, which influence cell proliferation, differentiation, or survival;
and organisation of the cytoskeleton. PAK proteins can regulate the activity
of the mitogen-activated protein kinase (MAPK) signalling cascade by direct
phosphorylation of Raf-1 and MEK (King et
al., 1998; Sun et al.,
2000
). Recently, it has also been shown that PAK6 associates with
the Androgen Receptor (AR) and translocates to the nucleus to repress
AR-mediated transcription (Yang et al.,
2001
). Different PAK proteins can also exert pro- and
anti-apoptotic effects (reviewed by Jaffer
and Chernoff, 2002
). A function of PAK proteins in regulation of
the cytoskeleton was first implicated by localisation of PAK1 to polymerised
actin (Sells et al., 1997
).
The morphological changes that have been attributed to different PAK proteins
include formation of lamellipodia, filopodia and membrane ruffles, as well as
the disassembly of focal adhesions (reviewed by
Bagrodia and Cerione, 1999
;
Daniels and Bokoch, 1999
;
Jaffer and Chernoff, 2002
).
Activated PAK4 can also induce loss of cell adhesion and anchorage-independent
growth, characteristic features of oncogenic transformation. Expression of
PAK4 is elevated in many tumour cell lines
(Callow et al., 2002
;
Qu et al., 2001
).
Localisation of PAK proteins to distinct cellular compartments appears to
be an important mechanism to control their function in the reorganisation of
the cytoskeleton. For example, activated Cdc42 causes the redistribution of
PAK4 to the Golgi compartment followed by actin polymerisation
(Abo et al., 1998).
Drosophila D-PAK colocalises with actin structures present in leading
edge cells, which elongate in the process of dorsal closure during
embryogenesis (Harden et al.,
1996
; Harden et al.,
1999
). Localised activation of D-PAK in the growth cone of
photoreceptor cell axons appears to be controlled by membrane recruitment of
D-PAK through binding to the Nck homologue Dock and by the guanine exchange
factor Trio, which controls Rac1 activity
(Hing et al., 1999
;
Newsome et al., 2000
).
We have analysed the function of the Drosophila group II PAK
protein Mbt in photoreceptor cell morphogenesis. In contrast to D-PAK
mutations, mutations in mbt do not cause axonal guidance defects, but
result in the frequent absence of photoreceptor cells in the eye and other
neurones in the brain (Hing et al.,
1999; Melzig et al.,
1998
). This suggests a role of Mbt in cell proliferation,
differentiation or cell survival. The Drosophila eye develops from a
monolayer epithelium: the eye-antennal imaginal disc
(Wolff and Ready, 1993
). As is
typical for polarised epithelial cells, contacts between cells in the
developing eye are mediated by adherens junctions (AJs), specialised membrane
structures that also play important roles in separating apical and basolateral
membrane domains and in coordination of cell shape changes (reviewed by
Tepass et al., 2001
).
Photoreceptor cells undergo massive morphological changes during terminal
differentiation. We show that Mbt localisation at AJs is required for normal
photoreceptor cell development. Mbt preferentially binds to GTP-loaded Cdc42.
A structure-function analysis revealed the importance of the Cdc42 binding
domain and the kinase domain for the in vivo function of Mbt. Besides
regulation of Mbt kinase activity, binding of Cdc42 to Mbt is required to
recruit Mbt to AJs. Our results provide evidence for a role of Mbt as a
downstream effector of Cdc42 in photoreceptor cell morphogenesis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of a Mbt antiserum
A mbt cDNA-fragment coding for amino acids 71-349 was amplified by
PCR and cloned as a EcoRI/XhoI fragment 3' to the
His-tag encoding sequences of the pET-28c expression vector (Novagen).
Expression and purification of the protein was carried out according to the
manufacturer's protocol. The purified protein was used for immunisation of
rabbits by a commercial supplier. The specificity of the antiserum was tested
by immunohistochemistry and western blot analysis.
Immunhistochemistry, histology and microscopy
Eye imaginal discs from third instar larvae or pupae were stained as
described by Gaul et al. (Gaul et al.,
1992), except that Cy3 or Alexa488-conjugated secondary antibodies
were used. The following primary antibodies were used: rabbit anti-Mbt
(1:500), mouse anti-Elav (1:10, provided by E. Hafen), mouse anti-Armadillo
(1:50, E. Knust), mouse anti-Crumbs (1:10, E. Knust), rabbit anti-Canoe
(1:500, D. Yamamoto), rabbit anti-Discs large (1:1000, A. Wodarz), rat
anti-mCD8 (1:100, Caltag) and anti-HRP-FITC (1:500, Cappel). Eye discs were
mounted in VectaShield and analysed with a LeicaTCS confocal microscope. Image
processing was carried out with the AMIRA software (TGS). For histological eye
sections, heads were fixed with OsO4 as described previously
(Basler et al., 1991
) and
embedded in EPON. Eye sections (1 µm) were stained with Toluidine
Blue/borax solution for microscopy.
Genetics, clonal analysis
Transgenic lines were generated by injecting Qiagen-purified plasmid DNA
into w1118 embryos. To analyse whether the wild-type or
the mutated mbt transgenes can rescue the mbtP1
eye phenotype, males carrying the appropriate UAS:mbt transgene were
crossed to mbtP1/mbtP1; Gal4:238Y/Gal4:238Y
females. Eye imaginal discs from the male progeny were dissected and analysed
by immunohistochemistry. Adult males were scored for rescue of the
mbtP1 eye phenotype. For each mbt construct, at
least two independent insertion lines were tested. The
UAS:Cdc42G12V and UAS:Cdc42T17N flies
were provided by Liqun Luo. To analyse the effect of
Cdc423 and Cdc424 mutations (flies
provided by Richard Fehon) on Mbt localisation, the mosaic analysis with a
repressible cell marker (MARCM) system was used
(Lee and Luo, 1999).
Homozygous Cdc423 or Cdc424 mitotic
clones were induced 72 hours after egg laying by two 60 minutes heat shocks at
37°C of animals of the following genotype: Cdc423/4,
FRT19A/tubP-Gal80, hs-Flp, FRT19A; GMR-Gal4/UAS:mCD8-GFP. Clonal tissue
was visualised by staining eye imaginal discs with an anti-mCD8 antibody.
Cell culture, Immunoprecipitation and western blot analysis
Human embryonic kidney (HEK) 293 cells were grown at 37°C in 5%
CO2 in Dulbecco's modified Eagle's medium (DMEM, Invitrogen)
containing 10% foetal calf serum (FCS), 1% penicillin-streptomycin and 1%
L-gutamine. For transient transfections, 6x105 HEK293 cells
were seeded in six-well plates and transfected 24 hours later with 2 µg of
the indicated DNA using the PolyFect reagent (Qiagen). Twenty-four hours after
transfection, cells were serum starved for 36 hours in DMEM medium containing
0.3% FCS, harvested in PBS and lysed at 4°C for 30-40 minutes in lysis
buffer (25 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA 10% glycerol,
0,1% NP-40 supplemented with 5 µg/ml Antipain, 1 µg/ml Aprotinin, 0,5
µg/ml Leupeptin, 0,7 µg/ml Pepstatin, 100 µg/ml PMSF). Total protein
lysate (100-250 µg) was incubated with 8 µl of a monoclonal anti-HA
antibody (clone12CA5) for 10 minutes, followed by incubation with
protein-G-Agarose beads (Roche) for 2 hours. Proteins were resolved by
SDS-PAGE and after transfer on nitrocellulose membranes probed with an anti-HA
antibody (1:1000, Roche) or a 1:2000 dilution of the anti-Myc antibody (clone
9E10, Santa Cruz). Proteins were detected using the ECL kit (Amersham
Pharmacia Biotech).
Protein kinase assay
HEK293 cells were grown after transfection in DMEM containing 10% FCS for
24 hours and then grown at low serum conditions (0.3% FCS) for additional
20-24 hours. After immunoprecipitation, the protein-G-Agarose beads were
washed twice with lysis buffer, once with 0,5 M NaCl and twice with kinase
buffer (20 mM HEPES pH 7.6, 20 mM MgCl2, 10 mM
ß-glycerophosphate, 20 mM p-nitrophenylphosphate). The kinase reaction
was performed in kinase buffer containing 0,5 mM Na3VO4,
2 mM DTT, 50 µM ATP, 5 µCi [-32P]ATP together with 5
µg myelin basic protein (MBP, Sigma) as a substrate for 30 minutes at
30°C. Proteins were visualised after SDS-PAGE by autoradiography.
GST Pull-down assay
The coding region of Cdc42 was cut out of the pcDNA3-myc-Cdc42
construct with EcoRI/NotI and cloned into the pGEX vector
(Amersham). The GST-Cdc42 fusion protein was expressed and
glutathion-sepharose purified according to standard procedures. After elution
with 100 mM glutathione, the protein was dialysed with 50 mM Tris pH 7.5
GDP/GTP loading of the fusion protein and the pull-down assay were performed
as described previously (Pandey et al.,
2002) with cell lysates transiently expressing HA-tagged Mbt.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To analyse the function of Mbt during eye development, we generated a
polyclonal antiserum and stained eye-antennal imaginal discs from third instar
larvae and pupae. Differentiation of the cells that comprise the single eye
units (ommatidia) occurs in a step-wise fashion and is initiated in the
morphogenetic furrow, which moves across the eye disc from posterior to
anterior (Wolff and Ready,
1993). Staining of third-instar eye-imaginal discs with the Mbt
antiserum revealed an accumulation of the Mbt protein at apical membrane sites
of the photoreceptor cells as soon as they become recruited to the ommatidial
clusters and initiate differentiation (Fig.
2A). Low levels of Mbt protein were detected at the membranes of
undifferentiated cells. Staining was completely absent in eye discs derived
from mbtP1 mutant larvae
(Fig. 2B), demonstrating the
specificity of the antiserum and confirming our previous notion that
mbtP1 is a complete loss-of-function allele
(Melzig et al., 1998
). To
determine the subcellular localisation of Mbt more precisely, we co-stained
eye discs with anti-Armadillo (Arm, Drosophila ß-Catenin)
antibodies, a marker for adherens junctions (AJs). Staining for both largely
overlaps in the photoreceptor cells (Fig.
2C,E). From apical to basal cross sections it became evident that
Mbt is less abundant in the most apical domain of Arm expression in the
photoreceptor cells.
|
The final architecture of the ommatidia is established during pupal
development and is accompanied by major morphological changes
(Longley and Ready, 1995;
Wolff and Ready, 1993
). At 37%
of pupal development (p.d.), the apical domains of the photoreceptor cells
have involuted. Thus, the apical domains of the photoreceptors point towards
the centre of the ommatidial cluster. After involution, the apical membranes
of the photoreceptor cells start to expand to form the rhabdomeres. Each
rhabdomere is surrounded by the stalk membrane, which connects it to the
zonula adherens. As shown by anti-Arm staining
(Fig. 3), the AJs span the
whole proximal-to-distal length of the photoreceptors at 50% p.d. Mbt remains
colocalised with Arm at AJs of pupal photoreceptor cells at different stages
of their development (Fig. 3).
Higher levels of Mbt expression can also be seen in the future bristle cells,
whereas cone and pigment cells express low levels of Mbt. A 3D reconstruction
of a wild-type ommatidial cluster stained with anti-Arm and anti-Mbt
antibodies shows the colocalisation of both proteins at AJs of the
photoreceptor cells along the whole proximodistal length
(Fig. 3). In summary, these
data provide evidence that Mbt is localised at AJs of photoreceptor cells from
the initial recruitment to their final differentiation.
|
Mutations in mbt interfere with photoreceptor cell
morphogenesis
The observed phenotypes in mbtP1 eyes could result from
a defect in cell proliferation, photoreceptor cell recruitment or
differentiation. To determine whether mbt mutations affect
recruitment or early neuronal differentiation of photoreceptor cells,
wild-type and mbtP1 third instar larval eye discs were
stained with an antibody against the neuronal differentiation marker Elav.
Only rarely did mbtP1 ommatidia contain fewer
Elav-positive cells than wild-type clusters
(Fig. 2G,H). This result was
confirmed by using HRP as an independent differentiation marker (data not
shown). This suggests that a failure in recruitment of photoreceptor cells is
not the major cause of the mbt phenotype.
The specific localisation of Mbt at AJs prompted us to look for AJ defects
in mbtP1 third instar and pupal eye imaginal discs with an
anti-Arm antibody. In third instar eye discs, the AJs of the developing
photoreceptor cells appear disorganised. Frequently, the AJs extend laterally
(Fig. 2D,F). The AJ defects
become much more pronounced at pupal stages
(Fig. 3). At 37% p.d., AJs fail
to extend in proximodistal direction. At 50% p.d., AJs are fragmented and form
patchy and disorganised structures. To verify these results and to exclude the
possibility that mbtP1 disturbs only Arm localisation
without affecting AJs, mbtP1 eye imaginal discs were
co-stained with anti-Canoe antibodies as an independent AJ marker
(Matsuo et al., 1999). Canoe
and Arm remain colocalised in mbtP1 eye discs (data not
shown). In addition, we stained wild-type and mbtP1 pupal
eye discs with antibodies against the apical determinant Crumbs (Crb) and the
Discs large (Dlg) protein, which is a marker for septate junctions in
epithelial cells (reviewed by Tepass et
al., 2001
). Recently it has been shown that Crumbs is essential to
maintain AJ integrity during photoreceptor cell morphogenesis and is localised
at the stalk membrane between AJs and the rhabdomeres
(Izaddoost et al., 2002
;
Pellikka et al., 2002
).
Compared with wild-type ommatidia, Crb
(Fig. 4A,B) and Dlg
(Fig. 4C,D) are de-localised in
mbtP1 mutant cells. In summary these data suggest that Mbt
function is required in the developing photoreceptor cells to undergo their
morphological changes.
|
Mbt interacts with Cdc42
To gain insight into the molecular mechanisms that control Mbt function, we
next tested the binding of Mbt to Rho-type GTPases. Group I PAKs have been
shown to interact via the p21-binding domain (PBD) with GTP-loaded Rac and
Cdc42 but not with Rho, whereas the group II PAK proteins PAK4 and PAK5
preferentially bind to GTP-bound Cdc42 (Abo
et al., 1998; Dan et al.,
2002
; Pandey et al.,
2002
).
Myc-tagged versions of the Drosophila homologues of Cdc42, Rac1 and Rho1 were co-expressed with HA-tagged Mbt in HEK293 cells. Co-immunoprecipitation experiments revealed a nearly exclusive binding of Cdc42 to Mbt (Fig. 5A). Rac1 showed only a very weak interaction whereas no binding of Rho1 to Mbt was detected. The specificity of the interaction between Cdc42 and Mbt was tested by mutation of two conserved histidine residues in the PBD (Fig. 5C) to leucine (MbtH19,22L). The mutant Mbt protein was unable to bind to Cdc42 (Fig. 5A). Thus, the interaction between Cdc42 and Mbt is indeed mediated by the PBD. To determine whether activation of Rho-type GTPases influence binding to Mbt in vivo, the constitutively activated variants Cdc42G12V, Rac1G12V and RhoG14V were co-expressed with Mbt in HEK293 cells. Cdc42G12V showed an enhanced interaction with Mbt when compared to wild-type Cdc42 (Fig. 5A). This result indicated that only the active, GTP-bound form of Cdc42 binds to Mbt. To verify this result, Cdc42 was expressed as a GST-fusion protein in bacteria and used in pull-down experiments upon loading with GDP or GTP. Mbt selectively binds to GTP-loaded Cdc42 but not to unloaded or GDP-loaded Cdc42 (Fig. 5B). Thus, the preference for binding GTP-bound Cdc42 appears to be a common feature among group II PAKs.
|
Regulation of Mbt kinase activity in vitro by Cdc42 binding
The PBD of group I PAKs is C-terminally flanked by the kinase inhibitory
domain (KID). Binding of activated Cdc42 and Rac relieves the inhibitory
influence of the KID on PAK kinase activity
(Buchwald et al., 2001;
Chong et al., 2001
;
Lei et al., 2000
). In
addition, group II PAKs share significant sequence homology C-terminal to the
PBD, but the sequences differ significantly from the group I PAK KID
(Fig. 5C).
To analyse the influence of Cdc42 binding on Mbt kinase activity we used
the Cdc42 binding-deficient MbtH19,22L construct. A second Mbt
construct used in this study bears a mutation in the kinase domain. This
mutation (T525A), located in the linker region between subdomains VII and
VIII, corresponds to the T777A mutation in the Saccharomyces
cerevisiae PAK protein Ste20p and has been found to disrupt
autophosphorylation and catalytic activity of Ste20p
(Wu et al., 1995). HEK293
cells were transfected with HA-tagged wild-type or the presumptive kinase-dead
version of Mbt and the immunopurified protein complexes were incubated with
kinase buffer and [
-32P]ATP together with myelin basic
protein (MBP) as a substrate. Compared with wild type Mbt, the T525A mutation
strongly reduced autophosphorylation and substrate phosphorylation
(Fig. 5D). Co-expression of
Cdc42 with Mbt did not increase autophosphorylation or MBP phosphorylation
when compared with cells transfected with Mbt alone. Importantly,
co-expression of Mbt and the constitutively activated Cdc42G12V
construct slightly reduced rather than enhanced the ability of wild-type Mbt
to phosphorylate MBP. Conversely, the Cdc42-binding defective
MbtH19,22L protein showed a moderate increase of MBP
phosphorylation independent of co-expression with Cdc42 or
Cdc42G12V (Fig. 5D).
Autophosphorylation was not affected by removal of the Cdc42-binding site.
These results fit with previous observations that kinase activity of PAK4, 5
and 6 is not upregulated upon Cdc42 binding, whereas deletion of the PBD can
lead to enhanced kinase activity (Abo et
al., 1998
; Pandey et al.,
2002
; Yang et al.,
2001
). Thus, group II PAKs appear to differ from group I PAKs in
their mechanism to regulate kinase activity.
Requirement of the Cdc42 binding domain and the kinase domain for Mbt
function in vivo
In order to test the requirement of the Cdc42-binding domain and the kinase
domain for Mbt function in vivo, we expressed wild-type or mutated Mbt
proteins during eye development in the absence of the endogenous Mbt protein.
Northern blot analysis (Melzig et al.,
1998) and antibody staining
(Fig. 2B) indicated that
mbtP1, which carries a P-element insertion in the protein
encoding sequence, is a complete loss-of function allele. Previously we have
shown that Gal4:238Y (Tettamanti et al.,
1997
)-driven expression of a mbt cDNA in the brain is
sufficient to rescue the mbtP1 brain phenotype
(Melzig et al., 1998
). We
found that Gal4:238Y is also expressed in the eye-antennal imaginal disc in a
manner that closely resembles the expression pattern of the endogenous Mbt
protein (see below). Consistent with this observation, the eye phenotype of
mbtP1 flies was completely rescued by Gal4:238Y-driven
expression of the wild-type mbt cDNA
(Fig. 6A). By contrast, the
Cdc42-binding deficient MbtH19,22L protein was unable to rescue the
mbtP1 eye phenotype
(Fig. 6C), whereas the
kinase-defective MbtT525A construct partially rescued the
mbtP1 eye phenotype
(Fig. 6B).
|
In summary, these experiments have verified the importance of the Cdc42-binding domain and the kinase domain for the in vivo function of Mbt during eye development. The partial rescue ability of the MbtT525A construct indicated that some functions of Mbt are independent of kinase activity. The differences observed in the rescue ability of the kinase defective MbtT525A and the Cdc42 binding-deficient MbtH19,22L proteins also suggested that Cdc42 binding to Mbt influences Mbt function in a kinase-independent manner. One possibility we investigated was the proper localisation of the Mbt protein to AJs.
Localisation of Mbt depends on Cdc42
Group II PAKs lack the N-terminal binding site for the Nck/Dock adaptor
protein, which could provide a link to membrane-bound proteins. To investigate
whether the Cdc42-binding domain is responsible for the observed localisation
of Mbt to AJs, we expressed wild-type and mutated mbt cDNAs with the
Gal4:238Y driver line in a mbtP1 mutant background and
analysed the subcellular localisation of the corresponding Mbt proteins in
pupal eye discs. The expression pattern of the transgenic, non-mutated Mbt
protein in the eye imaginal disc closely resembled the expression pattern of
the endogenous Mbt protein. High levels of transgenic Mbt accumulate at the
AJs of the developing photoreceptor cells
(Fig. 7A-C). Consistent with
the complete rescue of the adult mbtP1 eye phenotype
(Fig. 6A), no morphological
abnormalities were observed when pupal eye discs were stained with anti-Mbt
(Fig. 7A) or anti-Arm
(Fig. 7B) antibodies. An
identical localisation pattern was observed when the kinase-defective
MbtT525A protein was expressed in the mbtP1
mutant background (Fig. 7D-F),
indicating that eliminating kinase activity does not influence the subcellular
distribution of the Mbt protein. However, as revealed by co-staining with an
anti-Arm antibody, the transgenic MbtT525A protein only partially
rescued the AJs defects of mbtP1 animals (compare with
Fig. 3). The AJs extend to some
degree in proximal-to-distal direction but still do not have a regular
architecture (Fig. 7D-F). This
result correlates with the partial rescue observed in adult eyes
(Fig. 6B). By contrast, the
Cdc42 binding-deficient MbtH19,22L protein did not accumulate at
AJs but instead was distributed within the cytoplasm
(Fig. 7G). Anti-Arm staining
revealed that the MbtH19,22L protein is unable to rescue the AJs
defects seen in mbtP1 mutant eye discs
(Fig. 7H,I). Thus, there is an
absolute requirement of the Cdc42 binding domain for localisation and function
of the Mbt protein during eye development. To exclude that the failure of the
MbtH19,22L protein to localise at AJs is not a secondary effect of
the mbtP1 phenotype itself, we also expressed the
MbtH19,22L protein in a wild-type background
(Fig. 7K-M). Although
endogenous and transgenic Mbt protein cannot be distinguished in this case,
two observations were made. First, the MbtH19,22L protein did not
cause any obvious AJs defects when expressed in a wild-type background
(Fig. 7L). Second, Mbt protein
was found at AJs and in the cytoplasm (Fig.
7K). Because no cytoplasmic Mbt protein was detected upon
expression of the non-mutated Mbt protein in a wild-type background (data not
shown), we conclude that it is the MbtH19,22L protein, which
localises in the cytoplasm.
|
To show that Cdc42 and not another protein bound to the PBD of Mbt is
responsible for localisation of Mbt to AJs, we wanted to analyse animals that
either lacked Cdc42 function or ectopically expressed mutated versions of the
Cdc42 protein. Because removal of Cdc42 function causes lethality
(Genova et al., 2000),
homozygous mutant Cdc423 or Cdc424
cell clones were generated using the MARCM system
(Lee and Luo, 1999
). Only
those cells that are homozygous for the Cdc42 mutation express the
membrane localised mCD8 marker. Most of the Cdc423 or
Cdc424 clones obtained in the eye disc contained only a
few mCD8-positive cells. Consistent with previous findings that Cdc42
mutant cells can initiate their differentiation into photoreceptor cells
(Genova et al., 2000
), the
majority of mCD8-positive (Cdc42 mutant) photoreceptor cells analysed
extend an axon. In Fig. 8, an
apical to basal projection view of an anti-Mbt (A) and an anti-mCD8 (B)
stained eye-imaginal disc with a single Cdc42 mutant photoreceptor
cell is shown. From single apical sections
(Fig. 8D-F) and apical-to-basal
cross-sections (Fig. 8G-I) it
is evident that Mbt is localised at the apical side, whereas the mCD8 marker
labels the whole cell surface of the Cdc42 mutant photoreceptor cell,
including the axonal projection. Loss of Cdc42 function is accompanied by the
loss of apical Mbt protein.
|
Finally, we analysed the influence of the constitutively activated
(GTP-loaded) Cdc42G12V and of the dominant-negative (GDP-loaded)
Cdc42T17N protein (Luo et al.,
1994) on Mbt localisation. Because expression of these constructs
with the Gal4:238Y driver line resulted in embryonic lethality, the
eye-specific GMR-Gal4 driver line was used. Consistent with our finding that
Mbt only binds to GTP-loaded Cdc42, expression of Cdc42T17N had
only minor effects on Mbt localisation and the AJs morphology
(Fig. 8K,L). By contrast,
Cdc42G12V caused a dramatic change in the Mbt and Arm expression
pattern (Fig. 8M,N). Mbt
accumulates at membrane sites of all cells. Arm expression can only be seen at
early stages of photoreceptor cell recruitment, indicating that
Cdc42G12V completely disrupts the integrity of AJs in the
developing photoreceptor cells.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One major difference between group I and group II PAKs is the regulation of
kinase activity. For group I PAK proteins it has been shown that binding of
GTP-bound Cdc42 or Rac releases the inhibitory effect of the KID on catalytic
activity (Buchwald et al.,
2001; Chong et al.,
2001
; Lei et al.,
2000
). The lack of an obvious KID in group II PAKs
(Fig. 5C) is reflected by their
distinct biochemical properties. In contrast to group I PAKs, a slightly
reduced rather than enhanced kinase activity is observed upon co-expression of
Mbt and a constitutively active variant of Cdc42 in serum starved cells. A
Cdc42 binding-deficient Mbt protein showed enhanced kinase activity in vitro.
Similar results have been reported for other group II PAKs. Kinase activity of
PAK4 was not further enhanced upon co-transfection of activated Cdc42 but
deletion or mutation of the PBD of PAK4 and PAK6 resulted in enhanced kinase
activity (Abo et al., 1998
;
Callow et al., 2002
;
Yang et al., 2001
).
From these data, the question remains of what role activated Cdc42 plays in
regulating the functions of group II PAKs. Our genetic studies have verified
the importance of the kinase domain and the PBD for the in vivo function of
Mbt. Despite increased kinase activity in vitro, a construct lacking the PBD
was unable to rescue the mbtP1 mutant phenotype in the
eye. However, a kinase-dead Mbt protein partially rescued the
mbtP1 phenotype. This indicated that Cdc42 binding to Mbt
fulfils some additional essential functions that are independent of kinase
activity. Localisation studies showed that one major function of the PBD is to
recruit Mbt specifically to adherens junctions. These data are also supported
by our observation, that localisation of a PBD-deficient Mbt protein to the
cellular membrane by fusing it to a general membrane targeting sequence is not
sufficient to restore the wild-type function of the protein (T. R. and D. S.,
unpublished). We therefore propose that Cdc42 has a dual function: specific
recruitment of Mbt to AJs and regulation of the catalytic activity of Mbt. The
importance of proper targeting of PAK proteins to distinct subcellular
compartments for their in vivo function is also evident from other studies.
PAK4 recruitment to Golgi membranes by activated Cdc42 is dependent on an
intact PBD (Abo et al., 1998).
Activated Rac and Cdc42 also promote the relocalisation of a recently
described group II PAK protein in Xenopus laevis, X-PAK5, from
microtubule networks to actin-rich regions
(Cau et al., 2001
). In the case
of group I PAKs, autophosphorylation of the Nck and PIX SH3 domain binding
sites has been suggested as a mechanism to control cycling between different
cellular compartments (Chong et al.,
2001
; Zhao et al.,
2000
). Also, D-PAK function in the photoreceptor axons and growth
cones is dependent on the interaction with the Drosophila Nck
homologue dreadlocks (Dock), which binds to the tyrosine phosphorylated axon
guidance receptor DSCAM through its SH2 domain
(Hing et al., 1999
;
Schmucker et al., 2000
).
Rho GTPases are important regulators of the actin cytoskeleton and are
involved in many developmental processes that require morphological changes of
epithelial and neuronal cells (reviewed by
Luo, 2000;
Van Aelst and Symons, 2002
).
Each GTPase regulates a diverse range of effector molecules and thus induces
multiple defects when misregulated. For this reason, it is difficult to
reconcile all the data obtained with the loss-of-function Cdc42 alleles and
the various ectopically expressed Cdc42 variants
(Eaton et al., 1995
;
Genova et al., 2000
;
Harden et al., 1999
;
Luo et al., 1994
;
Riesgo-Escovar et al., 1996
),
but a number of conclusions can be drawn with respect to the function of Mbt
and D-PAK. First, activated Cdc42 has an effect on the levels of D-PAK
accumulating at the dorsal most ends (leading edge) of epidermal cells
flanking the amnioserosa (Harden et al.,
1999
). This is consistent with our result that overexpression of
activated Cdc42 in the eye disc leads to accumulation of Mbt at the membrane.
Second, the mbt and the Cdc42 mutant phenotypes in the eye
display some similarities. In the eye disc, cells devoid of endogenous Cdc42
or Mbt function can initiate their differentiation into photoreceptor cells
(Genova et al., 2000
)
(Fig. 2H). In the adult eye,
loss of Cdc42 function also causes the loss of photoreceptor cells and defects
in rhabdomere morphology of the remaining photoreceptor cells (D. S. and T.
R., unpublished). At first sight the similarities in the loss-of-function
phenotypes contradict the biochemical data implying a negative role for Cdc42
in kinase activation. In addition, the Cdc42 binding-deficient Mbt protein,
despite enhanced kinase activity in vitro, does not cause visible phenotypes
in the eye even when expressed in a wild-type background
(Fig. 7). There are several
possibilities to reconcile the data. In all cases, Mbt is either not present
or not localised to AJs. As discussed above, some functions of Mbt might be
independent of kinase activity. Alternatively, the moderate increase in kinase
activity of the MbtH19,22L protein might not be sufficient to
induce dominant phenotypes. A more detailed analysis of Mbt kinase activity in
vivo requires the identification of physiological substrates.
The described roles of PAK proteins and RhoGTPases in regulating the actin
cytoskeleton and the results presented in this study imply that Mbt localised
at AJs mediates signals to the cytoskeleton to ensure proper photoreceptor
cell morphogenesis. Because Mbt interacts only with GTP-loaded Cdc42,
recruitment of Mbt to AJs would require localised Cdc42 activation. Although
we have no direct evidence so far for a selective Cdc42 activation at AJs in
photoreceptor cells, studies in mammalian epithelial cell lines have
demonstrated Cdc42 activation by the AJ protein E-cadherin
(Kim et al., 2000). In
addition, the molecular link between Mbt and the actin cytoskeleton remains to
be defined. Similar than reported for PAK4, the Drosophila homologue
of LIMK could provide a link between Mbt and the actin cytoskeleton
(Dan et al., 2001a
;
Ohashi et al., 2000
). Based on
the similar mutant phenotype, the PDZ domain protein Canoe could be another
interaction partner of Mbt. Canoe is localised at AJs and the mammalian
ortholog Afidin has been shown to bind to actin filaments
(Matsuo et al., 1999
).
Unravelling the precise molecular functions of Mbt in cell morphogenesis
awaits the identification of interaction partners and physiological
substrates.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abo, A., Qu, J., Cammarano, M. S., Dan, C., Fritsch, A., Baud,
V., Belisle, B. and Minden, A. (1998). PAK4, a novel effector
for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and
in the formation of filopodia. EMBO J.
17,6527
-6540.
Bagrodia, S., Bailey, D., Lenard, Z., Hart, M., Guan, J. L.,
Premont, R. T., Taylor, S. J. and Cerione, R. A. (1999). A
tyrosine-phosphorylated protein that binds to an important regulatory region
on the Cool family of p21-activated kinase-binding proteins. J.
Biol. Chem. 274,22393
-22400.
Bagrodia, S. and Cerione, R. A. (1999). Pak to the future. Trends Cell Biol. 9, 350-355.[CrossRef][Medline]
Bagrodia, S., Taylor, S. J., Jordon, K. A., van Aelst, L. and
Cerione, R. A. (1998). A novel regulator of p21-activated
kinases. J. Biol. Chem.
273,23633
-23636.
Basler, K., Christen, B. and Hafen, E. (1991). Ligand-independent activation of the sevenless receptor tyrosine kinase changes the fate of cells in the developing Drosophila eye. Cell 64,1069 -1082.[Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Buchwald, G., Hostinova, E., Rudolph, M. G., Kraemer, A.,
Sickmann, A., Meyer, H. E., Scheffzek, K. and Wittinghofer, A.
(2001). Conformational switch and role of phosphorylation in PAK
activation. Mol. Cell. Biol.
21,5179
-5189.
Callow, M. G., Clairvoyant, F., Zhu, S., Schryver, B., Whyte, D.
B., Bischoff, J. R., Jallal, B. and Smeal, T. (2002).
Requirement for PAK4 in the anchorage-independent growth of human cancer cell
lines. J. Biol. Chem.
277,550
-558.
Cau, J., Faure, S., Comps, M., Delsert, C. and Morin, N.
(2001). A novel p21-activated kinase binds the actin and
microtubule networks and induces microtubule stabilization. J. Cell
Biol. 155,1029
-1042.
Chong, C., Tan, L., Lim, L. and Manser, E.
(2001). The mechanism of PAK activation. Autophosphorylation
events in both regulatory and kinase domains control activity. J.
Biol. Chem. 276,17347
-17353.
Dan, C., Kelly, A., Bernard, O. and Minden, A.
(2001a). Cytoskeletal changes regulated by the PAK4
serine/threonine kinase are mediated by LIM kinase 1 and cofilin.
J. Biol. Chem. 276,32115
-32121.
Dan, I., Watanabe, N. M. and Kusumi, A. (2001b). The Ste20 group kinases as regulators of MAP kinase cascades. Trends Cell Biol. 11,220 -230.[CrossRef][Medline]
Dan, C., Nath, N., Liberto, M. and Minden, A.
(2002). PAK5, a new brain-specific kinase, promotes neurite
outgrowth in N1E-115 cells. Mol. Cell. Biol.
22,567
-577.
Daniels, R. H. and Bokoch, G. M. (1999). p21-activated protein kinase: a crucial component of morphological signaling? Trends Biochem. Sci. 24,350 -355.[CrossRef][Medline]
Eaton, S., Auvinen, P., Luo, L., Jan, Y. N. and Simons, K. (1995). CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium. J. Cell Biol. 131,151 -164.[Abstract]
Galisteo, M. L., Chernoff, J., Su, Y. C., Skolnik, E. Y. and
Schlessinger, J. (1996). The adaptor protein Nck links
receptor tyrosine kinases with the serine-threonine kinase Pak1. J.
Biol. Chem. 271,20997
-21000.
Gaul, U., Mardon, G. and Rubin, G. M. (1992). A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase. Cell 68,1007 -1019.[Medline]
Genova, J. L., Jong, S., Camp, J. T. and Fehon, R. G. (2000). Functional analysis of cdc42 in actin filament assembly, epithelial morphogenesis, and cell signaling during Drosophila development. Dev. Biol. 221,181 -194.[CrossRef][Medline]
Harden, N., Lee, J., Loh, H. Y., Ong, Y. M., Tan, I., Leung, T., Manser, E. and Lim, L. (1996). A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures. Mol. Cell. Biol. 16,1896 -1908.[Abstract]
Harden, N., Ricos, M., Ong, Y. M., Chia, W. and Lim, L.
(1999). Participation of small GTPases in dorsal closure of the
Drosophila embryo: distinct roles for Rho subfamily proteins in epithelial
morphogenesis. J. Cell Sci.
112,273
-284.
Hing, H., Xiao, J., Harden, N., Lim, L. and Zipursky, S. L. (1999). Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila. Cell 97,853 -863.[Medline]
Izaddoost, S., Nam, S. C., Bhat, M. A., Bellen, H. J. and Choi, K. W. (2002). Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature 416,178 -183.[CrossRef][Medline]
Jaffer, Z. M. and Chernoff, J. (2002). p21-Activated kinases: three more join the Pak. Int. J. Biochem. Cell Biol. 34,713 -717.[CrossRef][Medline]
Kim, S. H., Li, Z. and Sacks, D. B. (2000).
E-cadherin-mediated cell-cell attachment activates Cdc42. J. Biol.
Chem. 275,36999
-37005.
King, A. J., Sun, H., Diaz, B., Barnard, D., Miao, W., Bagrodia, S. and Marshall, M. S. (1998). The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338. Nature 396,180 -183.[CrossRef][Medline]
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible neurotechnique cell marker for studies of gene function in neuronal morphogenesis. Neuron 22,451 -461.[Medline]
Lei, M., Lu, W., Meng, W., Parrini, M. C., Eck, M. J., Mayer, B. J. and Harrison, S. C. (2000). Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch. Cell 102,387 -397.[Medline]
Longley, R. L. and Ready, D. F. (1995). Integrins and the development of three-dimensional structure in the Drosophila compound eye. Dev. Biol. 171,415 -433.[CrossRef][Medline]
Lu, W., Katz, S., Gupta, R. and Mayer, B. J. (1997). Activation of Pak by membrane localization mediated by an SH3 domain from the adaptor protein Nck. Curr. Biol. 7, 85-94.[Medline]
Lu, W. and Mayer, B. J. (1999). Mechanism of activation of Pak1 kinase by membrane localization. Oncogene 18,797 -806.[CrossRef][Medline]
Luo, L., Liao, Y. J., Jan, L. Y. and Jan, Y. N. (1994). Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8,1787 -1802.[Abstract]
Luo, L. (2000). Rho GTPases in neuronal morphogenesis. Nat. Rev. Neurosci. 1, 173-180.[CrossRef][Medline]
Manser, E., Loo, T. H., Koh, C. G., Zhao, Z. S., Chen, X. Q., Tan, L., Tan, I., Leung, T. and Lim, L. (1998). PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell 1,183 -192.[Medline]
Matsuo, T., Takahashi, K., Suzuki, E. and Yamamoto, D. (1999). The Canoe protein is necessary in adherens junctions for development of ommatidial architecture in the Drosophila compound eye. Cell Tissue Res. 298,397 -404.[CrossRef][Medline]
Melzig, J., Rein, K. H., Schaefer, U., Pfister, H., Jaeckle, H., Heisenberg, M. and Raabe, T. (1998). A protein related to p21-activated kinase (PAK) that is involved in neurogenesis in the Drosophila adult central nervous system. Curr. Biol. 8,1223 -1226.[Medline]
Newsome, T. P., Schmidt, S., Dietzl, G., Keleman, K., Asling, B., Debant, A. and Dickson, B. J. (2000). Trio combines with Dock to regulate Pak activity during photoreceptor axon pathfinding in Drosophila. Cell 101,283 -294.[Medline]
Ohashi, K., Hosoya, T., Takahashi, K., Hing, H. and Mizuno, K. (2000). A Drosophila homolog of LIM-kinase phosphorylates cofilin and induces actin cytoskeletal reorganization. Biochem. biophys. Res. Commun. 276,1178 -1185.[CrossRef][Medline]
Pandey, A., Dan, I., Kristiansen, T. Z., Watanabe, N. M., Voldby, J., Kajikawa, E., Khosravi-Far, R., Blagoev, B. and Mann, M. (2002). Cloning and characterization of PAK5, a novel member of mammalian p21-activated kinase-II subfamily that is predominantly expressed in brain. Oncogene 21,3939 -3948.[CrossRef][Medline]
Pellikka, M., Tanentzapf, G., Pinto, M., Smith, C., McGlade, C. J., Ready, D. F. and Tepass, U. (2002). Crumbs, the Drosophila homologue of human CRB1/RP12, is essential for photoreceptor morphogenesis. Nature 416,143 -149.[CrossRef][Medline]
Pirone, D. M., Carter, D. E. and Burbelo, P. D. (2001). Evolutionary expansion of CRIB-containing Cdc42 effector proteins. Trends Genet. 17,370 -373.[CrossRef][Medline]
Qu, J., Cammarano, M. S., Shi, Q., Ha, K. C., de Lanerolle, P.
and Minden, A. (2001). Activated PAK4 regulates cell adhesion
and anchorage-independent growth. Mol. Cell. Biol.
21,3523
-3533.
Riesgo-Escovar, J. R., Jenni, M., Fritz, A. and Hafen, E. (1996). The Drosophila Jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate specification in the eye. Genes Dev. 10,2759 -2768.[Abstract]
Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E. and Zipursky, S. L. (2000). Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell 101,671 -684.[Medline]
Sells, M. A., Knaus, U. G., Bagrodia, S., Ambrose, D. M., Bokoch, G. M. and Chernoff, J. (1997). Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Curr. Biol. 7,202 -210.[Medline]
Sun, H., King, A. J., Diaz, H. B. and Marshall, M. S. (2000). Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak. Curr. Biol. 10,281 -284.[CrossRef][Medline]
Tepass, U., Tanentzapf, G., Ward, R. and Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35,747 -784.[CrossRef][Medline]
Tettamanti, M., Armstrong, J. D., Endo, K., Yang, M. Y., Furukubo-Tokunaga, K., Kaiser, K. and Reichert, H. (1997). Early development of the Drosophila mushroom bodies, brain centres for associative learning and memory. Dev. Genes Evol. 207,242 -252.[CrossRef]
Van Aelst, L. and Symons, M. (2002). Role of
Rho family GTPases in epithelial morphogenesis. Genes
Dev. 16,1032
-1054.
Wolff, T. and Ready, D. F. (1993). Pattern formation in the Drosophila retina. In The Development of Drosophila melanogaster (ed. M. Bate and A. Martinez-Arias), pp.1277 -1326. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Wu, C., Whiteway, M., Thomas, D. Y. and Leberer, E.
(1995). Molecular characterization of Ste20p, a potential
mitogen-activated protein or extracellular signal-regulated kinase kinase
(MEK) kinase kinase from Saccharomyces cerevisiae. J. Biol.
Chem. 270,15984
-15992.
Yang, F., Li, X., Sharma, M., Zarnegar, M., Lim, B. and Sun,
Z. (2001). Androgen receptor specifically interacts with a
novel p21-activated kinase, PAK6. J. Biol. Chem.
276,15345
-15353.
Zhao, Z. S., Manser, E. and Lim, L. (2000).
Interaction between PAK and Nck: a template for Nck targets and role of PAK
autophosphorylation. Mol. Cell. Biol.
20,3906
-3917.