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INTRODUCTION |
p21-activated kinases
(PAKs)1 are serine/threonine
kinases that bind to and in some cases are catalytically activated by
Rac and Cdc42 GTPases (1, 2). They were shown to actively regulate the
actin cytoskeleton dynamics through a number of different targets. They
control cell fate commitment, by acting as upstream regulators during
cell cycle progression and apoptosis (3-7). Their function in the
control of neurite outgrowth and growth cone collapse (8-11) may
explain the discovery that mutations in the human PAK3 gene are
responsible for X-linked mental retardation (12, 13).
Mammalian PAK1 to -6 as well as Drosophila,
Caenorhabditis elegans, and yeast PAKs (2, 14) all share a
highly conserved CRIB domain inside the GTPase-binding
domain/p21-binding domain (PBD). That domain is also conserved among
most other Cdc42 and Rac effectors (15). Mammalian PAKs of subgroup
I (PAK1/
, PAK2/
, and PAK3/
) share another feature, which is an
autoinhibitory domain (AID) partially overlapping the CRIB domain (2,
16, 17). The AID is composed of a dimerization segment, an
inhibitory switch segment (IS), and a kinase-inhibitory segment (18).
Intermolecular interactions were shown to occur between the conserved
AID and the catalytic domain of two different kinase monomers,
resulting in inactive PAK dimers under resting conditions (18, 19).
The main mechanism of PAK catalytic activation results from their
interaction with the GTP-bound form of the GTPases Rac and Cdc42
(1). GTPase binding to the PAK CRIB domain induces a conformational
change of the inhibitory switch in the AID, which interrupts the
interaction between the AID and the catalytic domain and leads to
monomeric PAK in an opened conformation (18). Autophosphorylation at
several sites in both the regulatory domain and in the kinase domain
then occurs and maintains the open conformation, allowing kinase
activation (20). However, membrane recruitment of PAK is also required
for its activation (21), and to date several potential mechanisms have
been described. Indeed, under resting conditions, PAK1 to -3 interact
with the Nck adapter that upon activation links tyrosine kinase
receptors and brings PAK to the membrane (for a review, see Ref. 22).
However, PAKs also form stable complexes with the guanine exchange
factors for Rac and Cdc42 of the PIX/Cool family, and these
interactions are not only closely involved in targeting PAK to the
membrane but also in regulating its kinase activity (23-25).
Despite sharing high sequence identity, PAK1, -2, and -3 differ in
pattern of expression. Indeed, PAK2 is ubiquitously expressed and is
activated by proteolytic cleavage during apoptosis and plays a
cytostatic role (26). On the other hand, PAK1 and PAK3 are restrained
to the nervous system (27) and are regulated at the time of the growth
cone guidance (10). But to date, no specific functions were attributed
to either neuronal PAK1 or PAK3.
Here we report the first characterization of a splice variant of the
mouse PAK3 gene we isolated from brain mRNA. This new cDNA
contains an alternatively spliced exon (exon b) of 45 bp that is
totally conserved in humans. The 15 amino acids encoded by this exon
are located in the regulatory domain, inside the inhibitory switch.
This PAK3b isoform is abundant in the brain and possesses high basal
kinase activity that is not further activated by active Cdc42 proteins.
Moreover, the alternatively spliced exon b inhibits
interactions with active GTPases Rac and Cdc42. Our results strongly
indicate that PAK3b may act through original pathways designed for
specific functions in neuronal signaling.
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EXPERIMENTAL PROCEDURES |
cDNA Cloning of PAK3 Isoforms--
An RT-PCR-based approach
was used to isolate cDNAs encoding PAK3 isoforms from adult mouse
brain mRNA. Based on published mouse PAK3 coding sequence (15, 28),
a set of two primers containing engineered BamHI and
XbaI restriction sites (underlined) was designed to
amplify the open reading frames of mouse PAK3 (primer set 1:
5'-CCGGATCCAGGATCTGACAGCTTGGATAACG-3' and
5'-GGCTCTAGACTAACGGCTACTGTTCTTAATTGC-3'). A high
fidelity Pfu polymerase (Promega) was used for the
amplification, and the PCR products were cloned into TOPO 2.1 vector
(Invitrogen) following the manufacturer's instructions and sequenced.
The isoform without insertion was named PAK3a, and the isoform
containing the insert was named PAK3b. The accession numbers for the
mouse PAK3a and PAK3b are AJ496262 and AJ496263, respectively. The
amino acid numbering of PAK3 used in this article corresponds to the
coding sequence of mammalian PAK3 protein.
Plasmid Construction--
The BamHI/XbaI
fragments of the PAK3a and PAK3b cDNAs were cloned into the
pcDNA3 vector in order to obtain HA-tagged PAK3 expression plasmids
named pHA-PAK3a and pHA-PAK3b, respectively. Mutants and constructs
were prepared from pHA-PAK3a and pHA-PAK3b plasmids with Pfu
polymerase, using procedures based upon the QuickChange protocol
(Stratagene) and confirmed by sequencing. In order to obtain a
constitutive activation of the kinase, the oligonucleotide set
(5'-CCTGAGCAAAGTAAACGAAGCGAGATGGTGGGAACTCCCTAT-3' and
5'-CCAATAGGGAGTTCCCACCATCTCGCTTCGTTTACTTTGCTC-3') was used for
site-directed mutagenesis of the threonine residue 421 to a glutamate
residue, as initially described (29, 30). In order to obtain dead
kinase mutants, the oligonucleotide set
(5'-CTGGACAAGAGGTGGCCATACTGCAGATGAACCTTCAACAGCAGCC-3' and
5'-GGCTGCTGTTGAAGGTTCATCTGCAGTATGGCCACCTCTTGTCCAG-3') was used to
change the lysine residue 297 to leucine, located in the putative ATP
binding site of the kinase domain of PAK3. The constitutively active
mutant was named pHA-PAK3a-ca, and kinase-dead mutants were named
pHA-PAK3a-kd and pHA-PAK3b-kd.
The PBD domains of PAK3 (amino acids
Lys65-Lys136) (28) were amplified with the
oligonucleotide set (5'-CCGGATCCAAAGAGCGCCCAGAGATCTC-3' and
5'-GACTCGAGTCATTTCTGGTTGTTGACCGTTTC-3'). The
BamHI/XhoI fragments were digested and subcloned
into pGEX-6P vector (Amersham Biosciences), leading to pGST-PBD-PAK3a
and pGST-PBD-PAK3b constructs. The AIDs of PAK3 isoforms (amino acids
His78-Ser146) (17) were amplified with the
oligonucleotide set (5'-GCGGATCCCATACGATTCATGTGGGT-3' and
5'-GCCTCGAGTTAACTTTTATCTCCTGACGT-3'). Fragments were
BamHI/XhoI subcloned into pGEX-6P (Amersham
Biosciences), leading to pGST-AID-PAK3a and pGST-AID-PAK3b constructs.
The vector pEGFP-C3 was purchased from Clontech.
Plasmids expressing GFP-fused active GTPases (RhoAG14V, RifQ77L,
RhoGG12V, Rac1G12V, Cdc42G12V, TC10Q75L, TCLQ79L, and ChpQ89L) were a
generous gift from P. Fort (CNRS, CRBM, Montpellier). The GST-Rac1wt,
GST-Cdc42wt, and GST-Cdc42V12 prokaryotic expression plasmids were
previously described (3).
Expression Analysis by RT-PCR--
RNAs from human brain or from
adult mouse tissues were prepared and reverse transcribed as previously
described (31). Expression of the new PAK3b isoform was characterized
from mouse or human brain cDNA using an exon b-specific sense
primer and a species-specific reverse primer, leading to a mouse
set (primer set 2: 5'-CCAGATCTCTATGGCTCACAG-3' and
5'-GGAGGAGCCAAAGGAGGTTC-3') or a human set (primer set 3: 5'-CCAGATCTCTATGGCTCACAG-3' and 5'-GGAGGGGCCAATGGAGGC-3'), giving rise
to a 275-bp fragment. A mouse multiple tissue cDNA panel was used
to analyze the expression of PAK3 isoforms by using the exon b-specific
primer set 2 as mentioned above and the mouse PAK3 isoform primer set 4 (5'-TGAGCAATGGGCACGACTAC-3' and 5'-CTTGGTGCAATGACAGGCGG-3'), giving
rise to a 297-bp fragment. To confirm successful reverse transcription
and to normalize the samples, a 500-bp fragment of
-actin was
amplified in parallel using primer set 5 (5'-CCAACCGTGAAAAGATGACC-3' and 5'-AATTGAATGTAGTTTCATGGATG-3'). The PCR products were
separated on agarose gel and analyzed after ethidium bromide staining.
Purification of Recombinant Proteins--
GST-PBD-PAK3a,
GST-PBD-PAK3b, GST-AID-PAK3a, GST-AID-PAK3b, GST-Rac1wt, GST-Cdc42wt,
and GST-Cdc42V12 recombinant proteins were expressed in
Escherichia coli and purified on glutathione-agarose beads
as described by the manufacturer (Amersham Biosciences).
Antibodies--
Antibodies to HA (12CA5) and to GFP were
purchased from Roche Molecular Biochemicals. The anti-PAK3-N19 antibody
(N19) directed against the 19 N-terminal amino acids was purchased from
Santa Cruz Biotechnology, and the anti-phospho-PAK1
(Thr423), which also recognizes the
phospho-Thr421 of PAK3, was purchased from Cell Signaling
Technology.
A rabbit polyclonal antiserum was raised against the exon b-encoded
synthetic peptide PDLYGSQMCPGKLPE conjugated to keyhole limpet
hemocyanin (Sigma). Antibodies were affinity-purified by Ultralink
column chromatography (Pierce) after covalent attachment of the
corresponding peptide, following the manufacturer's instructions. For
Western blot analysis, protein samples were separated by 10% SDS-PAGE
and transferred to polyvinylidene difluoride membrane (Millipore).
Immunodetection was performed using the SuperSignal chemiluminescent
reagent (Pierce). Quantification of chemiluminescence was performed
after acquisition with a CDD camera (SynGene) and quantification
software (GeneSnap and GeneTools; SynGene).
Cell Culture and Transfections--
COS-7 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 2 mM glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin (Invitrogen). Plasmid DNA (10 µg) was
transfected into 3 × 106 COS-7 cells using the
electroporation method with an electroporator (Bio-Rad).
Immunoprecipitation and in Vitro Kinase Assay--
Transfected
cells were lysed as previously described (31). In some experiments, in
order to activate the kinase, cleared cell extracts from
PAK3-transfected cells were incubated with 5 µg of recombinant
Cdc42V12 in the presence of 25 µM ATP during 30 min at
room temperature. Extracts were then immunoprecipitated by incubating
with 4 µl of 12CA5 anti-HA antibody plus 40 µl of Pansorbin
(Calbiochem) for 3 h at 4 °C. After washing, aliquots of
immunocomplexes were subjected to immunoblotting to
ensure that PAK proteins were correctly expressed and immunoprecipitated.
For kinase reactions, immunoprecipitates were washed once more time in
the kinase buffer (25 mM HEPES, pH 7.4, 25 mM
MgCl2, 25 mM
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM orthovanadate). Immunoprecipitates were then incubated in the kinase buffer containing 20 µM ATP and 5 µCi of [
-32P]ATP
(Amersham Biosciences) for 20 min at 30 °C, in the absence or in the
presence of H2B (3 µg; Sigma) or MBP (3 µg; Invitrogen) as a
substrate. Boiling in SDS-Laemmli sample buffer stopped the reaction,
and the products were resolved by SDS-PAGE. The incorporation of
32P was quantified using a PhosphorImager (Amersham Biosciences).
For the kinase activity inhibition by AID, immunoprecipitated HA-PAK3a
protein was incubated with increasing concentrations of GST-AID-PAK3
proteins in the presence of Cdc42V12 and ATP for 30 min at 30 °C
before performing a kinase assay as previously described
(17).
GTPase Overlay, Pull-down Assay, and
Co-immunoprecipitation--
The overlay assay was done as previously
described by Faure et al. (3). Briefly, 1 µg of purified
recombinant GST alone or in fusion with PBD-PAK3a or PBD-PAK3b was
loaded onto a 12% SDS-polyacrylamide gel and transferred to
polyvinylidene difluoride membrane, and the overlay assay was performed
using recombinant purified wild-type GST-Cdc42wt or GST-Rac1wt loaded
with [
-35S]GTP (3). For overlay assays with
full-length PAK3 proteins, the same procedure was used from HA-PAK3
plasmid-expressing COS-7 cells. Briefly, COS-7 cells were transfected
with pHA-PAK3a, pHA-PAK3b, or mutants. The amount of PAK3 protein in
the immune complexes was first determined by Western blotting on an
aliquot. Samples containing equal amounts of PAK3 proteins were used
for the overlay assay as described before. Quantification of the
binding of [35S]GTPases was done using a PhosphorImager.
Pull-down assays were performed as described by Vignal et
al. (32). Briefly, COS-7 cells were transfected with constructs expressing GFP alone or fused to the constitutive active mutants of
GTPases of the Rho/Rac/Cdc42 family. 30 h later, cells were washed
in cold phosphate-buffered saline and lysed in 50 mM
Tris-HCl, pH 7.5, 2 mM MgCl2, 1% Triton, 100 mM NaCl, 10% glycerol, and protein inhibitor mixture.
After 15 min on ice, lysates were cleared by centrifugation at
20,000 × g for 15 min. Cleared cell extracts were
incubated with 20 µg of GST, GST-PBD-PAK3a, or GST-PBD-PAK3b recombinant proteins, immobilized on glutathione beads, for 30 min at
4 °C. The beads were washed with the lysis buffer, and precipitated
proteins were analyzed by Western blotting, using the anti-GFP antibody.
Co-immunoprecipitation was performed as described by Reeder et
al. (33). Briefly, COS-7 cells were transfected with 5 µg of
active GFP-tagged Rac or Cdc42 along with 5 µg of HA-tagged PAK3a and
PAK3b constructs. Cell lysates in Robert's lysis buffer were
immunoprecipitated overnight with anti-HA antibodies (anti-HA affinity
matrix; Roche Molecular Biochemicals). Immune complexes were separated
by electrophoresis and transferred to polyvinylidene difluoride
membranes before Western blotting analysis with anti-PAK3 N19
antibodies to detect PAK3 isoforms or with anti-GFP antibodies to
detect co-precipitated Cdc42 or Rac1 proteins.
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RESULTS |
Identification of a New PAK3 cDNA--
In order to clone mouse
PAK3 cDNA, we performed RT-PCRs on mouse brain mRNAs and
subcloned and sequenced the PCR-amplified products. The sequence of the
mouse gene we isolated (accession number AJ496262) is slightly
different from published sequences (accession numbers U39738 and
AF082297) (15, 28). The main difference resides in Leu to Phe amino
acid substitution at position 271. A Phe residue is present as well in
the homologous position in human (AF068864) and rat (U33314) (34, 35). But most importantly, we found that some of the clones contained an
additional in-frame 45-bp sequence (Fig.
1A). Sequence of several clones generated by independent RT-PCR confirmed the presence of this
insert in certain PAK3 mRNA. Moreover, this sequence was identified
in several mouse expressed sequence tags (BE952172, AU08080098,
BB621877, and BE952177) (36). The isoform without insertion was named
PAK3a, and the isoform containing the insert was named PAK3b (accession
number AJ496263).

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Fig. 1.
Identification of the mammalian PAK3
alternatively spliced exon b. A, partial nucleotide and
predicted amino acid sequences of the new mouse PAK3 insert and
sequence alignment between mouse and human PAK3 isoforms at the border
of exons 2 and 3 (indicated with arrows). The
numbering refers to published sequences and starts at the
ATG/initiation codon of the mouse PAK3 gene. Exonic sequences are
indicated by capital letters, and intronic
sequences are indicated by lowercase letters. The
insert sequence is indicated in boldface letters.
B, RT-PCR analysis of the expression of the new PAK3b
isoform. Amplification reactions that were performed from mouse and
human brain RNAs (lanes 1) or no RNA as negative
control (lanes 2) using an exon b-specific primer
set were loaded on 2% agarose gels. Size markers are indicated in bp.
The lengths of amplified DNA fragments are in good agreement with the
expected size.
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Using the same approach, we then searched for this insert in different
mammalian species. To do so, human and mouse brain RNAs were reverse
transcribed and amplified using a specific forward primer for the 45-bp
insert and reverse species-specific primers, both designed to amplify a
275-bp fragment. The size of the DNA fragment that we amplified in both
species is in agreement with a 45-bp insertion as shown in Fig.
1B. An identical product was obtained from rat brain RNA
(data not shown). Finally, a BLAST search conducted in human genomic
data banks, using the 45-bp inserted sequence, demonstrated that this
sequence is strictly identical to a sequence lying on chromosome X in
position Xq22.3-q23 within the human PAK3 gene (34). The sequence is
located within the 5540-bp intron lying between exon 2 and exon 3 and
is flanked by 4342 and 1172 base pairs of intronic sequences at its 5'-
and 3'-end, respectively. The flanking sequences of the 45-bp insert are in good agreement with the consensus for donor and acceptor splicing sites (37). Altogether, these results demonstrate that the
45-bp insertion in mouse PAK3 cDNA defines a new alternatively spliced exon, henceforth referred to as exon b. We further show that
this alternatively spliced exon of PAK3 is strictly conserved in
different mammalian species.
Identification of the 68-kDa PAK3b Isoform--
Exon b translation
leads to a 15-amino acid in-frame addition that has no significant
identity with any sequence in the currently available databases.
Alignment of PAK3a, the previously characterized PAK3 isoform without
an alternatively spliced exon, with the different PAK3b sequences in
the region surrounding exon b is shown in Fig. 1A.
In order to specifically identify PAK3 isoforms, we developed
antibodies (IS-Eb) directed against the peptide encoded by the alternatively spliced exon b. To ensure the specificity of IS-Eb antibodies, COS-7 cells were transiently transfected with pHA-PAK3a (Fig. 2A, lane 2)
and pHA-PAK3b (Fig. 2A, lane 3)
constructs. Transfected cells were lysed 24 h later and further
analyzed in Western blots. In pHA-PAK3b transfected cells,
affinity-purified IS-Eb recognized a single 70-kDa protein but failed
to detect any band in pHA-PAK3a-transfected COS cells (Fig.
2A). In contrast, the N19-PAK3 polyclonal serum (as well as
anti-HA monoclonal antibodies; data not shown) recognized 67- and
70-kDa proteins in pHA-PAK3a- and pHA-PAK3b-transfected cells,
respectively (Fig. 2A). Indeed, these controls demonstrate
that IS-Eb antibodies are specific of the PAK3b isoform. Next, to
detect and identify PAK3 endogenous isoforms in adult mouse brain, we
performed immunoprecipitation of PAK3 proteins from brain lysate using
the N19-PAK3 serum that reacts with both isoforms. Immunoprecipitates
were further analyzed by Western blot and probed with both N19 and
IS-Eb sera. Two 65- and 68-kDa protein species were recognized with N19
antibodies, whereas the IS-Eb serum detected a unique 68-kDa species
(Fig. 2B). Thus, the latter 68-kDa/PAK3b species is
expressed in adult mouse brain at a significant and slightly lower
level than PAK3a. In summary, our results show that two different PAK3
isoforms, namely PAK3a and PAK3b, are expressed in adult mouse
brain.

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Fig. 2.
Immunodetection of the PAK3b isoform.
A, characterization of the IS-Eb antibodies. Control vector
(lanes 1), PAK3a (lanes 2),
and PAK3b (lanes 3) encoding plasmids were
transfected in COS-7 cells. Lysates were run on SDS-PAGE and analyzed
after Western blotting (WB) with antibodies against the
PAK3-N terminus region (N19) or the 15-amino acid PAK3 insert (IS-Eb).
Molecular masses are indicated in kDa on the left. As shown,
IS-Eb antibodies recognize specifically the PAK3b isoform.
B, PAK3a and -b isoforms were expressed in mouse brain.
Mouse brain extracts were immunoprecipitated with the N19-PAK3
antibodies and resolved by Western blotting with the N19 antibodies
(lane 1) or IS-Eb-specific antibodies
(lane 2). The arrows indicate specific
bands. The size difference between recombinant proteins in COS cells
and endogenous proteins in brain is due to the presence of a HA tag at
the amino-terminal extremity of the recombinant proteins.
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Expression--
These results raised the question of whether the
two PAK3 gene products PAK3a and PAK3b may display a narrow and
distinct tissue specificity. To address this question, we studied the
expression profile of mouse PAK3b by semiquantitative RT-PCR in various
adult tissues. Because PAK3 expression is known to be restricted to brain and, at a lower level, testis (35), we investigated PAK3 RNA
expression in different adult mouse tissues as well as in different
regions of the brain. First, equal amounts of first strand cDNA
were analyzed after normalization using
-actin (Fig. 3C). Then the tissue
distribution of both PAK3 mRNAs was investigated by RT-PCR
amplification of the carboxyl-terminal part of the regulatory domain,
since this domain is not subject to alternative splicing. Our results
demonstrate that PAK3 (a and b isoforms) RNAs are highly expressed in
the different parts of the adult mouse brain, with a slightly higher
expression in the spinal cord and in the midbrain. PAK3 is highly
expressed in testis and to a lower extent in heart and muscles. No
expression was detected in other tissues (Fig. 3A). In
parallel, the analysis of expression of the PAK3b isoform was performed
using an exon b-specific forward primer as shown in Fig. 3B.
PAK3b expression was detected in the different parts of the brain and
in the spinal cord, and, like PAK3a, PAK3b displays a slightly
higher expression in midbrain and spinal cord. Nonetheless, PAK3b
transcript distribution is narrower than PAK3a, since, unlike PAK3a, it
is not detected in testis. Whereas PAK3b tissue distribution is not
specific to any brain territory, it appears to be restricted to the
brain in the adult mouse.

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Fig. 3.
Semiquantitative RT-PCR analysis of mouse
PAK3 and PAK3b isoform expression on different tissues. RT-PCR was
performed from RNAs isolated from total brain (B), cerebral
hemisphere (CH), cerebellum (CB), midbrain
(MB), medulla (M), spinal cord (SC),
thymus (TH), liver (L), testis (T),
kidney (K), heart (H), spleen (S),
skeletal muscle (MU), and negative control (C).
Amplification was performed using specific primer sets for PAK3a and -b
(primer set 4, upper panel), PAK3b (primer set 2, middle panel), and -actin for normalization
(primer set 5, lower panel).
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Kinase Activity--
Since the alternatively spliced exon b is
located within the N-terminal regulatory domain of PAK3, we
investigated whether its expression might interfere with the intrinsic
kinase activity in an in vitro kinase assay (Fig.
4). To do so, COS-7 cells were transfected with plasmids coding for either wild type PAK3a
(a) and PAK3b (b) isoforms or for their
corresponding kinase-dead mutants that are mutated on their ATP binding
site (a-kd and b-kd) or for a constitutively
active mutant for PAK3a (a-ca) (see "Experimental Procedures"). 48 h after transfection, cell lysates were
incubated with or without recombinant active Cdc42 protein, and the
kinase activity was tested on the purified immune complexes in two
ways. First, PAK3 autophosphorylation was estimated in the absence of substrate (autoP), and second, kinase activity was tested by
measuring the phosphorylation of the histone H2B as a substrate
(H2B) in independent assays (Fig. 4, A and
B). Nevertheless, the addition of the H2B substrate in the
kinase assay did not significantly affect the capacity of PAK3 to
autophosphorylate (data not shown). The kinase activity was calculated
with reference to the Cdc42-activated PAK3a activity in each
condition.

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Fig. 4.
Kinase activity of PAK3 isoforms and PAK3
mutants. COS-7 cells were transfected with plasmids encoding for
wild type PAK3a (a) and PAK3b (b) or for
constitutively active kinase mutant PAK3a-ca (a-ca) or
kinase-dead mutants PAK3a-kd (a-kd) and PAK3b-kd
(b-kd). Cell lysates were incubated in the absence or
presence of the active protein Cdc42V12 and immunoprecipitated with
anti-HA serum. The immunoprecipitates were subjected to an in
vitro kinase assay in the absence or presence of H2B as a
substrate. Phosphorylated proteins were resolved by SDS-PAGE, and the
gel was autoradiographed. Western blotting with aliquots confirmed that
similar amounts of recombinant protein were present in each sample
(data not shown). A, a representative autoradiography from
autophosphorylation of PAK3 proteins in the absence of exogenous
substrate (upper panel) or from the
phosphorylation of H2B as a substrate (lower
panel). B, the histograms shown represent an
average of three independent experiments. Gels were scanned and
analyzed with an Amersham Biosciences PhosphorImager using
ImageQuant software. Results are expressed as percentage of the
Cdc42-activated PAK3a kinase activity in each experimental condition
(i.e. for autophosphorylation (upper
histograms) and for H2B phosphorylation (lower
histograms)).
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In agreement with previously published results (28, 35), we found that,
in the absence of GTPases, wild type PAK3a activity (a)
measured by autophosphorylation or toward H2B substrate was low and
weakly detectable and corresponds to the basal activity. As expected,
the kinase-dead mutants of both PAK3a (a-kd) and PAK3b
(b-kd) had no detectable kinase activity. The constitutively active (a-ca) mutant displayed a 16-fold increase in
autophosphorylation activity in the absence of GTPases. Surprisingly,
we reproducibly found no significant increase of the kinase activity of
this mutant toward H2B. Very interestingly, we found that wild type
PAK3b activity is high in resting cells with an average 25-fold
increase in activity compared with wild type PAK3a activity, as
measured by autophosphorylation. Note that the PAK3b isoform
phosphorylated H2B with a very high efficiency in resting cells. In
fact, as seen in Fig. 4, A and B, PAK3b basal
kinase activity is higher than the constitutive PAK3a-ca mutant
(compare a-ca with b).
We then analyzed the potential activation of PAK3a and PAK3b following
incubation with a recombinant constitutively active GST-Cdc42V12
protein. As expected, Cdc42 incubation strongly increased Pak3a kinase
activity (27-fold in autophosphorylation), whereas no activation was
detected for PAK3a-kd protein. The PAK3a-ca mutant is also
activated upon Cdc42V12 interaction, although its activity toward the
exogenous H2B substrate was repeatedly lower than the activity
developed by the activated wild type kinase (PAK3a). Interestingly, the
PAK3b isoform was not further activated by active Cdc42. The level of
the PAK3b activity was identical to that of Cdc42-activated PAK3a activity.
The GTPase-mediated activation of PAK is accompanied by several
autophosphorylation events (29), one of them being the phosphorylation of the threonine 421 that is implicated in activation (38). Thus, to
confirm that PAK3b is constitutively active in cells, we looked for its
autophosphorylation. Using a specific anti-phosphopeptide Thr423 (homologous to the Thr421 of PAK3), we
indeed detected the PAK3b but not the PAK3a isoform by Western blot
after immunoprecipitation of PAK3 from transfected COS-7 cells (data
not shown). This suggests that PAK3b is constitutively phosphorylated
on threonine 421 in resting cells. Altogether, these results show that
the PAK3b isoform possesses a high basal kinase activity, which is not
stimulated by active Cdc42 protein.
The AID of PAK3b Did Not Inhibit Kinase Activity--
The main
mechanism of PAK autoinhibition is the inhibitory interaction of the
N-terminal part of the protein with the catalytic domain. This
interaction is inhibited by phosphorylation of the Thr421
residue (17, 18, 39). As previously reported, the peptide, which
encompassed residues 78-146 in the AID, is sufficient to inhibit the
kinase activity of PAK (17). We thus investigated the respective
ability of the PAK3a AID and the modified PAK3b AID to negatively
regulate kinase activity of the wild type PAK3a (Fig.
5). AID-PAK3a (AID-3a) and AID-PAK3b
(AID-3b) (amino acids 78-146) were purified as recombinant GST fusion
proteins. The wild type PAK3a protein was immunoprecipitated from
transfected COS-7 cells and then incubated with increasing amounts of
recombinant GST-AID and active Cdc42 (Fig. 5). The AID-3a inhibited
PAK3a kinase activity with a curve of concentration dependence with an
IC50 of about 50 nM (0.1 µg). However, when
the same experiment was done with the AID-3b, no inhibition of the
kinase activity was detected even at a concentration of 500 nM (1 µg). Thus, the presence of the 15 amino acid insert
of PAK3b greatly impairs the inhibitory properties of the
autoinhibitory domain.

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Fig. 5.
The autoinhibitory domain of PAK3b does not
inhibit kinase activity. Increasing amounts (0.05-1 µg) of
recombinant GST-AID proteins of either PAK3a or PAK3b isoforms were
incubated with immunoprecipitated HA-PAK3a protein in the presence of
active Cdc42V12 and ATP. The reaction was then subjected to an in
vitro kinase assay in the absence or presence of MBP as a
substrate. Phosphorylated proteins were resolved by SDS-PAGE, and the
gel was autoradiographed. GST alone did not inhibit the kinase activity
of PAK3a (data not shown). A, a representative
autoradiography from autophosphorylation. Similar results were obtained
with MBP substrate (data not shown). B, the histogram shows
the average of three independent experiments on MBP phosphorylation by
PAK3a kinase in the presence of 1 µg of recombinant GST-AID-PAK3a
(3a) or GST-AID-PAK3b (3b). The basal activity
value (in the absence of Cdc42V12) was first subtracted from each
value. Results are expressed as kinase activity, relative to the
Cdc42-activated PAK3a kinase activity ( ).
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Interaction with GTPases--
We showed that PAK3b kinase activity
is not stimulated by active Cdc42. To test whether the 15-amino acid
PAK3b insert might impede PAK3b interaction with Rac and Cdc42, we
analyzed PAK3 isoforms binding to active GTPases (Fig.
6). GST fusions of PBD-PAK3a (PBD-3a), PBD-PAK3b (PBD-3b), and GST alone were
purified and tested for their ability to bind in overlay assays with
[
-35S]GTP-loaded Cdc42 and Rac1. As expected, Fig.
6A shows that the PBD-PAK3a binds both active Rac and Cdc42.
In contrast, the binding of PBD-PAK3b to the GTPases is dramatically
reduced. Indeed, we quantified PBD-3b binding from several independent
experiments and found that it was reduced to 9 and 0% with Cdc42 and
Rac1, respectively (Fig. 6A, lower
panel). We observed the same level of binding inhibition
using the entire regulatory domain (fragment 2-272) of PAK3 proteins
(data not shown). Thus, our results strongly suggest that the exon b
impairs the binding of the PAK3b isoform with Rac and Cdc42
GTPases.

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Fig. 6.
GTPase binding to PAK3 isoforms.
A, upper panels, GTPase binding of
PBD-PAK3a (PBD-3a) or PBD-PAK3b (PBD-3b)
proteins. 1 µg of purified recombinant protein GST alone or in fusion
with PBD-PAK3a or PBD-PAK3b was separated by SDS-PAGE, transferred to
polyvinylidene difluoride membranes, and overlaid with
[ -35S]GTP-loaded Cdc42 or Rac1. Lower
panel, quantification of the relative binding to the active
GTPases of PBD-3b compared with PBD-3a binding. These data were
averaged from three independent experiments. B, expression
and GTPase binding of wild type and mutant HA-PAK3 isoforms. COS-7
cells were transfected with plasmids encoding for PAK3a (a)
or PAK3b (b), for constitutively active kinase PAK3a mutant
(a-ca), and for kinase-dead mutants (a-kd) and
(b-kd). PAK proteins were immunoprecipitated, and the amount
of PAK proteins in the immune complexes was determined by Western
blotting (WB) on an aliquot (upper
panel). Samples containing equal amounts of PAK3 proteins
were separated by electrophoresis, transferred to polyvinylidene
difluoride membrane, and overlaid with
[ -35S]GTP-loaded Cdc42 or Rac1. The typical profile of
an autoradiography is shown (middle panel).
Quantification of interactions with Cdc42 (gray) and Rac
(black) from three independent experiments was averaged
(histogram, lower panel).
C, pull-down of active GTPase by PAK3 isoforms. COS-7 cells
were transfected with plasmids encoding GFP-tagged active GTPases. Cell
lysates were directly loaded (input i) or were
incubated with 20 µg of GST-PAK3-PBD3a (a) or
GST-PAK3-PBD3b (b). Retained proteins were run on SDS-PAGE
and were analyzed by Western blotting with antibodies against GFP
protein. A typical profile is shown. Lower panel,
quantification from three independent experiments was averaged after
acquisition of the chemiluminescence with a CDD camera. D,
in vivo interaction of active GTPases with PAK3 isoforms.
HA-tagged PAK3 isoforms were co-expressed into COS-7 cells with either
GFP as control or active GFP-tagged Cdc42 or Rac. Upper
panel, the presence of GFP-GTPases in the cell lysates was
controlled by anti-GFP immunoblotting. Middle panel, PAK3
proteins were immunoprecipitated (IP) using anti-HA
antibodies, and immune complexes were separated by electrophoresis and
transferred to polyvinylidene difluoride membranes. The amount of
immunoprecipitated PAK3 proteins was controlled using anti-PAK3 N19
immunoblotting. Lower panel, the presence of the
GFP-tagged Cdc42 or Rac protein in the immune complexes was revealed by
anti-GFP Western blotting. Data shown are representative of a typical
experiment.
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To confirm this result, we analyzed the interaction of wild type PAK3a
and PAK3b and the mutant PAK3a-ca, PAK3a-kd, and PAK3b-kd proteins
expressed in transfected COS-7 cells with
[
-35S]GTP-loaded Cdc42 and Rac1 GTPases (Fig.
6B). We verified by Western blot against PAK3 antibodies
that all PAK3 proteins were expressed and immunoprecipitated at similar
levels in the transient assay (Fig. 6B, upper
panel). As shown in Fig. 6B, lower
panel, GTP-Rac1 bound to PAK3a, PAK3a-ca, and PAK3a-kd
proteins with a similar efficiency, suggesting that the GTPase/PAK3a
interaction does not depend upon PAK3a kinase activity. In contrast,
the PAK3b isoform has a reduced capacity to bind to Rac. Similar
results were obtained with GTP-Cdc42. PAK3b binding to Cdc42 and Rac
corresponds to 52 and 39%, respectively, of PAK3a binding (Fig.
6B, histogram). We observed in several
independent experiments that PAK3b-kd always bound to a lesser extent
to Cdc42 and Rac than PAK3b. This indicates that the impaired PAK3b
binding to GTPases we observed is not a consequence of the high kinase
activity of PAK3b. These data confirm the results obtained with the
recombinant PBD-(65-136) or Nter-PAK3-(2-272) proteins, but
the inhibition of binding of PAK3b isoform was lower with the
full-length proteins than with recombinant shorter proteins.
To further demonstrate that the expression of exon b interferes with
the ability of PAK3 to interact with members of the Rho/Rac/Cdc42 family, we developed an approach based on pull-down assays of the
active GTPases with GST alone or with GST-PBD-PAK3a and GST-PBD-PAK3b proteins. COS-7 cells were transfected with either GFP alone or constitutively active (V12/V14) GFP-tagged GTPase mutants. Transfected cell lysates were incubated with the GST fusion proteins before pull-down (Fig. 6C). As expected, control GST protein alone
did not precipitate any GTPase (data not shown), and expressed GFP protein alone, as control, did not interact with any GST-PBD bait. RhoA
binds neither to GST-PBD-PAK3a, as initially reported (28, 35), nor to
GST-PBD-PAK3b. GST-PBD-PAK3a, indeed, interacts with Rac and Cdc42
(Fig. 6C). GST-PBD-PAK3b did not pull down RhoA either and
pulled down Rac and Cdc42 GTPases with a lower efficiency than
GST-PBD-PAK3a. GST-PBD-PAK3b consistently pulled down less Rac than
Cdc42 (Fig. 6C, histogram).
Finally, to assess the binding of the PAK3 isoforms to Cdc42 and Rac in
an in vivo environment, we performed co-immunoprecipitation assays. HA-tagged PAK3 isoforms were co-expressed into COS-7 cells with
either GFP as control or active GFP-tagged Cdc42 or Rac. PAK3 proteins
were then immunoprecipitated using anti-HA antibodies, and the presence
of the associated GTPase in the immune complexes was revealed by
anti-GFP immunoblot (Fig. 6D). We ensured that similar
levels of active Cdc42 and Rac were present in the cell lysates using
anti-GFP immunoblotting (upper panel) and
verified that PAK3a and PAK3b proteins were immunoprecipitated at
similar levels (middle panel).
Co-immunoprecipitation of active GTPases with HA-tagged PAK3 isoforms
is presented in the bottom panel. As expected,
the two PAK3 isoforms did not interact with control GFP protein, and
PAK3a isoform bound active Cdc42 and Rac, although with a lower
efficiency for the latter. In contrast, relative to PAK3a binding, the
efficiency of PAK3b interaction with Cdc42 and Rac was reduced to 55 and 32%, respectively. We always noticed that two species of Cdc42
were resolved in Western blot, after co-immunoprecipitation with PAK3.
This probably results from the cleavage occurring during the
immunoprecipitation procedure, since no cleavage was observed in the
cell lysate (upper panel). For binding
quantification, both bands were considered.
Taken together, our results demonstrate that the 15-amino acid insert
in PAK3b PBD impairs PAK3b ability to interact correctly with both Rac
and Cdc42.
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DISCUSSION |
In the present study, we have shown that the mouse PAK3 gene, as
well as the human gene, encodes two isoforms of 544 and 559 amino
acids. The two isoforms are generated by exon skipping of a short
sequence of 45 bp. The sequence of the alternatively spliced exon is
strictly conserved between mice and humans. Thus, we report here the
first characterization of an isoform for a member of the p21-activated
kinase family. The alternatively spliced exon codes for 15 amino acids
that do not possess any identity with known sequences and appear to be
unique for 3b. Thus, the PAK3b mRNA codes for a new isoform
identified as a 68-kDa protein that is only slightly less abundant than
the PAK3a isoform in adult mouse brain extract.
The PAK3b insert is located between Thr92 and
Gly93 of the sequence FT*GIP, which is highly conserved in
the PAK family and is present in the six mammalian PAK1-6 proteins and
in other orthologs of PAK, i.e. in Xenopus,
Drosophila, C. elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and
Candida albicans (17, 40). This sequence FT*GIP is located
immediately after the CRIB domain, in the overlapping region of the
GTPase-binding domain/PBD and of the AID. It adopts different
conformations depending on the interaction with the GTPases (18, 41,
42). Indeed, in absence of GTPase, this segment forms a
strand at
the extremity of a
hairpin that interacts with the
-helices of
the IS domain, packing it in a compact structure (18, 41). The
active GTPase interacts with the CRIB domain and with residues of the
IS domain, producing a modification of the structure, which allows the
G protein to contact the
hairpin and the
-helices of the IS
domain and which disrupts the inhibitory conformation. Previous
analysis of mutations in PAK sequences, which interfere with GTPase
binding and the interaction between the regulatory domain and the
catalytic domain, have demonstrated that the region where the PAK3b
15-amino acid insert lies is very critical for both GTPase interaction and regulation of the kinase activity. Indeed, we found that PAK3b possesses unique GTPase binding properties and kinase activity regulation.
Kinase Activity--
The PAK3b isoform clearly differs from the
constitutively active mutant T421E. Whereas the mutant PAK1-T423E has
only a weak constitutive kinase activity toward the substrate H2B, its
ectopic expression is sufficient to deregulate the cell cytoskeleton
network in fibroblasts (30, 43). Indeed, PAK3b basal kinase activity is
more elevated than the constitutively engineered T421E PAK3-ca mutant
and is not, unlike this latter, further activated upon Cdc42
interaction (30, 43). Since the kinase activity of PAK3a-ca and PAK3b
are not similarly regulated, it is likely that their biological roles
may differ as well. Interestingly, it was reported that the double
mutation of Ser422 and Thr423 stimulates PAK1
activity more than the Thr423 mutation (29). This
highlights the multistep mechanism of PAK activation and the difference
between autophosphorylation and phosphorylation of
exogenous substrates.
How could the presence of the 15-amino acid insert activate the kinase?
Mutations inside the autoinhibitory domain are known to suppress its
inhibitory properties (16, 17). It was previously reported that
mutations of the conserved residues Phe91,
Gly93, and Pro95, which are located near the
insertion site, lead to a high basal kinase activity that was not
further activated by active Cdc42 (44). Similar results were obtained
by a genetic approach in the S. pombe PAK1 protein, where
the mutation of the homologous residues Phe84,
Gly89, Glu90, Phe91,
Thr92, Trp98, and Leu102 leads to
the suppression of intramolecular interaction and activation of the
kinase (45). We have shown that the AID of the PAK3b isoform cannot
inhibit the PAK3 kinase activity. Thus, the 15-amino acid insert
probably modifies the inhibitory conformation of the IS and disrupts
this functional domain. Another hypothesis is that the alternatively
spliced exon inhibits the formation of dimers that is necessary to
allow transinhibition (19). The dimerization segment, as well as some
other regions including the IS segment, contributes to the
stabilization of dimers (19). Thus, it is possible that the insert of
PAK3b suppresses dimer formation and, by consequence, the associated
inactive state of the kinase. Another hypothesis is that the
deformation of the closed conformation of the regulatory domain allows
the direct phosphorylation of the crucial Thr421, which in
turn leads to the kinase activation (20, 39). Finally, another
possibility is that the insertion could promote the activation of the
kinase by one of these previously described mechanisms, which in turn
induces the autophosphorylation of two residues, Ser139 and
Thr421, leading to an inhibition of the closed conformation
(39, 46). We are currently investigating which of these mechanisms is implicated.
GTPase Binding--
We observed a drastic decrease of GTPase
binding for the PAK3b isoform. This result was obtained by overlay,
pull-down, and co-immunoprecipitation assays. The overlay test was done
with three different constructs, corresponding to the PBD domains
(fragment 65-136), to the amino-terminal regulatory domain (fragment
2-272), and to full-length proteins from PAK3a and PAK3b isoforms.
Pull-down experiments of constitutively active mutants of Cdc42 and Rac were performed with the GST-PBD proteins. Finally, the in
vivo GTPase interaction was measured after immunoprecipitation of
PAK3 proteins from co-transfected cells. The main result we obtained is
that the PAK3b isoform interacts significantly less with GTPases than
PAK3a. The binding of the PBD or the N-terminal region of PAK3b is
between 0 and 10% compared with that of PAK3a. On the other hand, the
interaction of the full-length PAK3b protein relative to PAK3a was
around 50% for Cdc42 and only 15% for Rac. In the same way, the
co-immunoprecipitation of Cdc42 with PAK3b was diminished to 45%,
whereas the co-immunoprecipitation of Rac was reduced to 32%. Thus, we
conclude that the presence of the insert decreases GTPase interaction.
The CRIB domain was identified as a conserved motif present in the
different target proteins for both Rac and Cdc42 (15). This domain is
necessary but not sufficient for a high affinity binding (47, 48). The
smallest fragment of PAK that binds small G proteins with high affinity
consists of residues Ile70-Lys173 (47). Some
residues, (PAK1 Met99, homologous to the PAK3
Ile94, and also the conserved residues Trp98,
Leu101, and Leu102), located in the proximity
of the insertion, are involved in interaction with the GTPase (41).
Particularly, the mutation of PAK1 Gly98/PAK3
Gly93 decreases the affinity of PAK for GTPases (17). Thus,
it may be possible that the insertion of the exon b changes the IS
structure and impedes GTPase interactions in a similar manner.
It was also reported that following activation, the full-length PAK3
protein does not bind active GTPases (1, 35). But the fact that neither
the kinase-dead mutant of PAK3b nor its amino-terminal domain devoid of
kinase activity was able to bind the GTPases strongly suggests that the
inability of PAK3b to bind GTPases is not a consequence of its high
basal kinase activity. On the contrary, the fact that the PAK3b-kd
mutant binds the active GTPases to a lesser extent than the wild type
PAK3b suggests that some autophosphorylation events may increase the
binding of PAK3b to Cdc42 by an as yet unknown mechanism.
Moreover, this region of GTPase binding that encompasses the CRIB
domain is also implicated in the selectivity of the interaction with
Rac versus Cdc42 (33, 49). Interestingly, the mutation of
the residue Phe91 of PAK3, homologous to the
Phe96 of PAK1, abolished Rac binding more strongly than
Cdc42 binding (33). Consistent with this, the binding of the PAK3b
isoform is more impeded for Rac than for Cdc42. One hypothesis could be that the presence of the exon b inside the regulatory domain can modify
the interaction with some GTPases of the Rac/Cdc42 family. We
tested this by pull-down experiments with different GTPases of the
Rho/Rac/Cdc42 family. We did not find any interaction with Rif and
RhoG, which are paralogs of RhoA (data not shown) (50, 51). However, we
found that three other members of the Rac/Cdc42 family, TC10, TCL, and
Chp (32, 52, 53), bind PAK3a more strongly than PAK3b with a ratio
identical to that of Cdc42 (data not shown). It is interesting to note
that whereas active Rac binds PAK3a with the same affinity as Cdc42,
Rac is only a weak activator of PAK3a (see Refs. 28 and 35; our
results). Thus, we can hypothesize that the different GTPases function
either to recruit and activate PAK3, as does Cdc42, or to only recruit PAK3 as does Rac. On the other hand, PAK3b brings in a new mechanism whereby Cdc42 could act only to recruit it, since it is already active.
These mechanisms are particularly interesting with regard to the
biological function of Rac and Cdc42 in neuritogenesis (54).
Another interesting question is whether the PAK3b isoform acts
independently of the Rac and Cdc42 GTPases. It was proposed that PAK
activation proceeds by several successive steps, the first one being
membrane recruitment by the Nck adaptor or the guanine exchange
factor of the PIX family and the second step being the
activation by GTPase or lipids (for a review, see Ref. 23). It may be
possible that PAK3b that possesses high basal kinase activity is
activated in a signaling pathway only by membrane recruitment, by a
mechanism independent from GTPases, and this remains to be tested.
However, it was shown that following activation, PAK
autophosphorylation prevents further binding to both Nck and Pix (55).
Thus, we can hypothesize that PAK3b may not interact with these two
partners and cannot be recruited by these pathways. Finally, PAK
mutants that have high kinase activity disrupt actin filaments and
focal adhesions (for a review, see Ref. 23) and cooperate with Ras,
Rac, and Rho GTPases in transformation or tumor cell line invasiveness
(56-58). This further indicates that PAK3b is unlikely to be
constitutively active in the nervous system and argues that a novel
unknown PAK regulatory mechanism independent of GTPases exists and
remains to be identified.
Finally, we report here the first characterization of an isoform for
the PAK3 kinase. This isoform is detected at similar levels in the
different parts of adult mouse brain. Future investigations of the
biological properties of each PAK3 isoform, in particular in neuronal
cells, may be of help to understand the function of PAK3 in the brain.
The PAK3 gene was found to be implicated in mental retardation twice
(12, 13). In one case, it is a missense mutation of the residue
Arg67, which probably disrupts GTPase binding, and in the
second case, the mutation is a nonsense mutation inside the kinase
domain generating a truncated protein. In both cases, these mutations
could affect the functions of the two isoforms. Whether the PAK3a or
PAK3b isoforms are implicated in mental retardation or in synaptic
plasticity is a crucial question. Our results indicate a complex role
for PAK3 isoforms in neuronal signaling.