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INTRODUCTION |
The p21-activated kinases
(PAKs)1 are ubiquitous
serine/threonine protein kinases that interact with the activated
GTP-bound forms of Cdc42 or Rac1 (1). Binding of Cdc42 or Rac to the N-terminal regulatory region of PAK is sufficient to stimulate in
vitro autophosphorylation and >100-fold activation of the kinase. PAKs are regulated by a variety of extracellular signals that impinge
on these small GTPases as previously reviewed (2-4). Four distinct PAK
isoforms are known in mammals; these are closely related to PAKs found
in worms and flies (1, 3, 5). Additional species like PAK4 are
Cdc42-associated but are not catalytically activated upon binding (6),
because they lack a regulatory kinase inhibitory domain (KID). This
inhibitory region flanking the Cdc42/Rac1 interaction/binding (CRIB)
region binds to and negatively regulates the catalytic domain (7-9).
The
PAK (2) isoform contains a caspase-sensitive site that results
in proteolytic activation of
PAK during apoptosis (10).
Mammalian PAKs seem to also play an important role in promoting
turnover of focal complexes (FCs) and actin stress fibers (11).
In vivo inhibition of PAK results in a failure of its upstream regulators Cdc42 or Rac1 to breakdown FCs and stress fibers
(7). These effects are in part mediated by PAK inhibition of myosin
light chain kinase (12), although it is also reported that PAK can
exert the opposite action of directly activating type II myosin light
chains (13). PAK is also capable of driving changes in cell morphology
resembling those elicited by Rac1 (14) mediated by its partner PIX, an
Rac1 quanine nucleotide exchange factor that promotes
lamellipodial formation (15). These activities apparently derive from a
requirement for PAK binding to PIX, which is in turn coupled to the
important signaling protein PI3K (16, 17).
Although basic proteins such as histone H4 and myelin basic protein
(MBP) are good substrates of PAK, it is unlikely these represent
bone fide targets. In vitro experiments indicate
that PAK is a "basic directed" kinase but is unusual in tolerating substrates with acidic residues at the
1 position of the substrate (18). PAK was recently identified as the key kinase in regulating Ser-338 of Raf1, and maximal Raf1 activation appears to be
PAK-dependent (19, 20). Inspection of this site reveals
that indeed the unusual PAK selectivity probably derives from the
flanking Asp-337. In addition to its effects on the actin cytoskeleton,
PAKs may play a role in disassembling intermediate filaments composed
of desmin (21). Studies on PAK as a protease-activated kinase (22) indicated that the catalytic domain needs to undergo
ATP-dependent autotransphosphorylation prior to conversion
to its active form (23). The behavior of PAK as a dimer in solution (9)
suggests that transphosphorylation of the kinase can be rapid. The
structure of the PAK catalytic domain·KID complex shows a
substantial interface between the two (9) consistent with a measured
Ki of 90 nM using PAK-(83-149)
(7).
In this study, we have investigated the role of autophosphorylation
sites in both Cdc42- and sphingosine-mediated PAK activation. Through
the use of PAK autophosphorylation site substitution mutants, we show
that these sites (which are largely conserved among the mammalian PAK
isoforms) play distinct roles. In particular, the activity of PAK is
controlled not only by modification of the "activation loop" but
also by changes in the KID. The latter thus facilitates
transphosphorylation within the kinase domain. Multiple phosphorylation
events cooperate in vivo to regulate both location and
activity of the kinase.
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MATERIALS AND METHODS |
Generation of Mutant PAK cDNAs--
- and
PAK
cDNAs were cloned into the mammalian expression vectors pXJ-HA or
pXJ-GST, which have been described previously (11, 16). These cDNAs
were excised and moved to pGEX 4T-1 for Escherichia coli
expression in the BL21 strain using the 5'-BamHI site and
3'-XhoI site. Autophosphorylation site mutants were
generated in these cDNAs using the QuikChange kit (Stratagene)
under the manufacturer's conditions. Mutants were confirmed by
sequencing of the relevant portion of the cDNA.
Cell Culture and Transient Transfection--
HeLa cells were
grown in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum to 50-80% confluency in 100-mm dishes. After starving
for 2 h, transient transfection was performed by incubation with a
complex containing 4 µg of DNA and 25 µl of DOSPER in 0.5 ml of
serum-free medium for 45 min, which was added to the cells in media
containing 1% fetal bovine serum. Cells were harvested 20 h later.
PAK Purification, Immunoprecipitation, and Kinase
Assays--
Cultured cells (100-mm plates) were harvested in 300 µl
of lysis buffer (25 mM HEPES, pH 7.3, 0.3 M
NaCl, 1.5 mM MgCl2, 0.5 mM EGTA, 20 mM
-glycerophosphate, 1 mM sodium vanadate,
0.5% Triton X-100, 5% glycerol, 0.5 mM
phenylmethylsulfonyl fluoride, and 1 µg/ml each of pepstatin,
leupeptin, and aprotinin). Extracts were incubated with anti-FLAG or HA
antibody for 2 h and then passed through a 30-µl column of
protein A-Sepharose. The beads were washed with 1 ml of
phosphate-buffered saline + 0.1% Triton then PAK activity assayed in
kinase buffer (50 mM HEPES, pH 7.3, 10 mM
MgCl2, 10 mM NaF, 2 mM
MnCl2, 1 mM dithiothreitol, 0.05% Triton)
containing 10 µCi of [
-33P]ATP and 0.2 mg/ml myelin
basic protein (MBP). The reaction was stopped after 15 min by adding
SDS sample/loading buffer. The relative levels of MBP phosphorylation
was quantified using a PhosphorImager (Molecular Dynamics).
Immunoprecipitated PAK levels were monitored by Western blotting.
Proteins were resolved on 9% SDS-polyacrylamide gels were transferred
to polyvinylidene difluoride membranes (PerkinElmer Life Sciences).
These were probed with affinity-purified rabbit anti-
PAK antibodies
as previously described (11).
The GST-PAK proteins were purified from cells lysates (as above), but
glutathione-Sepharose (Amersham Pharmacia Biotech) was used to trap the
protein, which was subsequently released either by addition of thrombin
(10 units/ml, 1-h room temperature) or with 2 column volumes of 10 mM glutathione in kinase buffer (without MBP). Purified
kinase was assessed by a modified Bradford assay (Bio-Rad) and stored
at
70 °C. The integrity of the PAK proteins was assessed by
SDS-PAGE. For each kinase assay 100 ng of purified PAK was incubated in
25 µl of kinase buffer under the conditions given above. Sphingosine
(Sigma) was dissolved in dimethyl sulfoxide (Me2SO)
at 10 mg/ml and stored at
40 °C. Kinase assays were performed either in kinase buffer + 20% Me2SO or using
sphingosine vesicles obtained by sonicating sphingosine (1 mg/ml) in
phosphate-buffered saline immediately prior to the experiment.
For phosphopeptide identification (Fig. 1A) 10 µg of
purified
PAK was incubated with 10 µg of GST/Cdc42·GTP
S in 50 µl of kinase buffer containing 5 µM
[
-33P]ATP at 32 °C for 10 min, followed by addition
of "cold" ATP to 500 µM and further incubation for 30 min to ensure complete autophosphorylation. Phosphoamino acid analysis,
tryptic digestion, HPLC analysis and peptide microsequencing are as
previously described (11).
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RESULTS |
Conserved Autophosphorylation Sites among Different PAK
Isoforms--
Cdc42-mediated
PAK activation in vitro is
accompanied by autophosphorylation of recombinant
PAK on 6 serine
residues in the N-terminal regulatory domain and 1 threonine residue
within the catalytic domain (11). The
PAK isoform was found to be phosphorylated on all equivalent sites, but additionally Ser-19 and
Ser-165 were phosphorylated (24). We were interested to test the other
PAK isoform (
PAK/PAK3) particularly with a view to identifying the
primary autophosphorylation events. The
PAK was purified from
transiently transfected COS7 cells and activated in the presence of
excess Cdc42·GTP
S (see "Materials and Methods") but limiting
ATP concentration; 2 µM kinase was incubated with 5 µM [
-33P]ATP (followed by
phosphorylation to completion in 500 µM ATP). Fig.
1A shows the profile of
HPLC-separated
PAK tryptic peptides: Radiolabeled residues
(asterisks) were then determined from 33P
release during the sequencing cycles. The results indicate that the
initial phosphorylation events in
PAK occur at residues Ser-50, Ser-139, and Thr-421, corresponding to Ser-57, Ser-144, and Thr-422 in
PAK (Fig. 1B). The limited number of autophosphorylation
sites identified in this analysis reflects differential rates of
autophosphorylation of the potential sites at limiting ATP
concentration: More weakly labeled peptides were not subjected to
further analysis.

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Fig. 1.
Conservation of PAK autophosphorylation
sites. A, purified PAK (10 µg) was activated
in vitro with excess Cdc42·GTP S in the presence of 5 µM [ -33P]ATP then to completion (see
"Materials and Methods") and subjected to in-gel tryptic digestion.
HPLC separation of peptides was as described previously (11). Cerenkov
counts detected for each peptide are displayed graphically, and the
sequence of the four major radiolabeled species was determined. A
portion of the material was then subjected to Edman cycles with
scintillation counting to determine the position of
[33P]phosphate in the peptide (asterisk).
Peptide 12 contained radiolabeled Ser-50, peptide 13 radiolabeled
Ser-139, and peptides 38/39 radiolabeled Thr-421. B,
schematic array of the major groups of autophosphorylation sites in
PAK. Note groups I and IV occur adjacent to the major binding sites for
PAK partners Nck and PIX. The primary sites of PAK
autophosphorylation occur flanking the CRIB/KID and in the kinase
domain.
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In
PAK as with
PAK, all the non-kinase autophosphorylation sites
are serine residues (see Fig. 1B); however,
PAK S19
(equivalent to
PAK T20) was found to be phosphorylated to the same
extent as
PAKS20 (24). We have recently shown the latter site to be important for the regulation of Nck SH3 binding (25). Because mutant
PAK(T422S) is activated normally by Cdc42-GTP
S, but no phospho-threonine is detected (Fig.
2A) is seems
PAK T20 is not an autophosphorylation site. This mutagenesis was performed in the
L404S background, which allows for recovery of E. coli
expressed kinase with low basal activity (11).

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Fig. 2.
Threonine 422 modification is not required
for autophosphorylation. A, thrombin-cleaved
GST/ PAK(L404S) proteins from E. coli and containing
either the T422S or T422A substitutions were purified. Each reaction
contained 2 µg of kinase with or without 4 µg of Cdc42·GTP S
(in 25 µl) and myelin basic protein (5 µg). The reaction was
initiated by addition of 10 µM [ -33P]ATP
and incubated for 15 min at 32 °C. The samples were analyzed by 12%
SDS-PAGE. The labeled PAK bands were excised and subjected to
phosphoaminoacid analysis as described previously (1). Note that the
T422S protein undergoes autophosphorylation but does not contain
phosphothreonine. B, the in vitro activity of
PAK and PAK T422A toward non-basic substrates. Purified GST
fusion protein substrates are shown in the right panel.
Myosin light chain (M) is phosphorylated by PAK in
vitro (40) and in vivo (13), GST-MEKK2-(1-120)
(MK, Ref. 41) is a particularly efficient substrate (our
unpublished observations), and the GIT1-(1-376) (G)
construct contains the N-terminal phosphorylated half of the substrate
normally found with PAK·PIX complexes (34). The kinase (0.5 µg) was incubated with 1 µg of Cdc42·GTP S and 5 µg of
substrate under standard conditions with [ -33P]ATP and
was quenched by addition of SDS sample buffer, followed gel
electrophoresis. The panel shows PAK autophosphorylation
(top) and substrate phosphorylation (bottom).
C, wild type PAK proteins from E. coli or
containing the substitutions as indicated (2-4) were purified and
assayed for activity as in part A. The activity toward MBP in each
experiment was normalized relative to unmodified PAK
(n = 3). The right-hand panels shows the
Coomassie Blue-stained PAK protein. The slower migrating species
reflect autophosphorylation of PAK.
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The Contribution of Thr-422 Phosphorylation to PAK
Activation--
Previous studies have shown that activation of the
catalytic domain requires phosphorylation of the residue equivalent to
PAK Thr-422, probably in "trans" (23, 26). However, although the
T422A mutant exhibited minimal activity toward substrate, it underwent
robust autophosphorylation with Cdc42-GTP
S (Fig. 2A).
Because myelin basic protein is not a known physiological PAK substrate
we carried out an additional experiment using three other substrates:
myosin light chain (13), the MEKK2-(1-290) regulatory region and
GIT1-(1-376), the latter 90-kDa protein is strongly phosphorylated in
the PAK·PIX·GIT complex (16). The T422A mutant was defective in
phosphorylation of all three substrates demonstrating that, although
Thr-422 phosphorylation is not required for the primary
(intramolecular) autophosphorylation events, it has a strong influence
on substrate recognition.
Mutants were also generated in the wild type
PAK background. In this
case the wild type PAK protein undergoes activation in the bacteria and
is recovered in an autophosphorylated state. Comparing the level of
activity of the T422A mutant with two other mutants substituted in the
N-terminal region (S144A/S149A and S198A/S203A), it was clear
that Thr-422 is not the only autophosphorylation site that can affect
activity (Fig. 2B). To test in vivo the notion that Thr-422 phosphorylation is not the sole determinant of PAK activity, the doubly substituted
PAK(L107F, T422A), which lacks a
functional kinase inhibitory domain (KID), was compared with
PAK(L107F). The kinase was expressed and immunoprecipitated from transiently transfected HeLa cells. As can be seen in Fig.
3A, and in contrast with
results using recombinant PAK, the T422A substitution reduces activity
of the
PAK(L107F) protein by only ~50%. Thus phosphorylation
events in the N terminus, as indicated by the significant mobility
shift of the
PAK(L107F, T422A), clearly can play roles in activating
PAK in vivo, although cellular phosphatases probably more
effectively down-regulate the double mutant as judged by the extent of
the mobility shift compared with
PAK(L107F).

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Fig. 3.
N-terminal phosphorylation events affect PAK
activity. A, wild type, PAKL107F or
PAKL107F/T422A FLAG-tagged proteins were purified from
COS-7 cells and quantified by Western analysis (right
panel). Activity was assayed on the anti-FLAG beads (25 µl) with
MBP and 10 µM [ -33P]ATP. The L107F/T422A
mutant exhibited slower mobility than wild type (WT) kinase
and a 25-fold higher activity. B, analysis of GST/PAK
protein purified from transiently expressing COS-7 cells. Sites in
PAK that are adjacent were mutated in pairs (i.e.
Ser-144/Ser-149 and Ser-198/Ser-203); other single substitutions are as
shown below each panel. Each lane contains 0.5 µg of purified protein
analyzed by SDS-PAGE. C, activity of various PAK proteins
as assessed by activity toward MBP. A typical profile of MBP
phosphorylation is shown, and the graph below plots the
average activity (of three measurements) for each construct. The PAK
cDNAs were transfected into COS-7 cells ± a vector encoding
Cdc42G12V. In each experiment the activity of wild type
kinase (WT) in the presence of Cdc42G12V was
taken as 100%. D, assays of PAK activity as in
C.
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A Conserved Serine in the KID Modulates PAK Activity--
To
investigate further the role of individual autophosphorylation sites,
additional
- and
PAK mutants were generated in which the
respective serine and threonine were changed to alanine. The closely
spaced sites in
PAK (Ser-144/Ser-149 and Ser-198/Ser-203) were
substituted together. We did not analyze Ser-21 (within the Nck binding
site), because PAK lacking this Nck-binding domain behaved identically
to full-length kinase in terms of in vitro activation (data
not shown). cDNAs encoding these mutants were transfected into HeLa
cells and the resulting GST/PAK fusion proteins purified and quantified
by Coomassie Blue staining (Fig. 3B). All the proteins were
recovered with similar yields, and no breakdown was detected on Western
analysis (not shown).
To analyze these PAK mutants during activation in vivo, the
cDNAs were co-transfected with an expression plasmid for
Cdc42G12V. Under these conditions, the smaller
Cdc42G12V protein was expressed in molar excess over PAK
(data not shown). Fig. 3C shows the activity of each mutant
expressed relative to that of wild type
PAK (activity designated
100%). The
PAKS57A and
PAKS198A/S203A
mutants were activated to the same extent as wild type
PAK, whereas
the purified S144A/S149A kinase was less active. Similarly purified
PAK mutants
PAKS50A and
PAKS200A were
as active as wild type, indicating these residues play no direct role
in kinase activation. The
PAKS139A and
PAKT421A proteins (equivalent to
PAK S144A or T422A)
were activated by Cdc42G12V to a lesser extent than wild
type implicating both these sites in the activation process. The double
PAKS144A/S149A mutant was somewhat less active than the
single
PAKS139A mutant (but
PAK does not contain a
site corresponding to
PAK Ser-149). These results are in agreement
with those obtained by in vitro activation of
PAK (see
Fig. 6). Thus a conserved serine within the KID (present in all
mammalian PAKs) is directly implicated in kinase activation.
Phosphorylation of the PAK Regulatory Region Blocks Its Inhibitory
Function--
The kinase inhibitory domain (KID), whose function
requires residues 83-149 (partially overlapping the p21-binding
domain), exhibits a Ki for PAK of 90 nM.
Indeed GST/
PAK-(83-149) or the larger Cdc42-binding-defective
GST/
PAK(S76P)-(1-250) proteins completely block activation of
-
and
PAKs by Cdc42·GTP
S (7). We therefore wished to test
directly whether phosphorylation of the inhibitor sequence could block
its function. Although GST/
PAK-(83-149) protein is phosphorylated
by active PAK (26), we found the phosphorylation reaction to be
inefficient with little subsequent effect on KID activity (not shown).
By contrast the larger GST/
PAK(S76P)-(1-250) protein was completely
phosphorylated by PAK (as assessed by gel shift in Fig.
4A), perhaps because the
autophosphorylation sites are essentially in a "native"
conformation. Fig. 4B shows that, although the untreated
inhibitor protein blocked
PAK activation, its phosphorylated
counterpart showed little activity. Thus phosphorylation of the PAK
N-terminal regulatory domain can suppress KID function. Inspection of
the recently solved structure of the
PAK kinase·KID complex (9)
confirms that phosphorylation of Ser-144 would sterically hinder
interactions at the interface (see "Discussion" and accompanying
Fig. 9A). This explains why phosphorylation of
PAK
Ser-144 or the corresponding
PAK Ser-139 plays a direct role in
modulating kinase activity.

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Fig. 4.
Phosphorylation of the PAK regulatory domain
blocks its inhibitory activity. A, immobilized
GST/ PAK-(1-250) containing the S76P substitution (7) was
phosphorylated by incubating with purified active PAK and 0.5 mM ATP. After an extensive (60 min) reaction, the kinase
was washed out, and GST/ PAK-(1-250) eluted with glutathione. The
shift in mobility indicates complete phosphorylation of the protein.
B, PAK activity (100 ng) was assayed with or without excess
(1 µg) PAK-(1-250)-S76P. The activation of PAK by
GTP S·Cdc42, which is blocked by PAK-(1-250)-S76P (compare
lanes 2 and 3), is essentially unaffected by the
phosphorylated inhibitor (lane 4).
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Sphingosine Activation of
PAK--
Both PAK1
autophosphorylation and "in gel" activity toward a p47phox peptide
have been reported to be stimulated by various sphingolipids (27).
Sphingosine is relatively selective, because closely related compounds
are ineffective in activating PAK, suggesting a specific interaction
with the kinase. Stimulation of
PAK activity in vitro was
half-maximal at ~500 µM sphingosine (Fig.
5A).
PAK activation was
poorer at higher concentrations of sphingosine (>1 mM).
This inhibitory effect of sphingosine at higher concentrations was
demonstrated when pre-activated
PAK was tested at 5 mM
sphingosine, indicating a direct inhibition of catalysis rather than
the activation process (Fig. 5B, right
panel).

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Fig. 5.
Sphingosine can both activate and inhibit
PAK. A, PAK (100 ng) was incubated in the presence
of sphingosine in 20% Me2SO and 5 µg of MBP at 32 °C
with 10 µM [ -33P]ATP. After 15 min an
equal volume of sample buffer was added and the reaction was analyzed
by 12% SDS-PAGE and autoradiography (top inset). The
quantified counts are plotted. B, sphingosine can act as
both activator and inhibitor: 0.5 mM sphingosine can
activate active PAK (left panel) but PAK pre-activated
with 0.5 mM sphingosine (+10 µM
"cold" ATP) is inhibited by 5 mM sphingosine when
assayed toward MBP (10 µM [ -33P]ATP).
C, both Cdc42 and sphingosine-mediated PAK activation are
blocked by addition of the KID. PAK (100 ng) was activated by
Cdc42·GTP S (250 ng) or sphingosine (100 µM) in the
presence and absence of 1 µg of GST PAK-(83-149) kinase inhibitory
domain (KID).
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Sphingosine- and Cdc42-mediated PAK Activation through Distinct
Sites--
The
PAK inhibitor polypeptide (residues 83-148) was
similarly effective in blocking both sphingosine- and Cdc42-mediated activation of
PAK in vitro (Fig.
6A). Thus sphingosine relieves autoinhibitory interactions within PAK by affecting the
inhibitor/kinase interface, although apparently it does not directly
bind at the interface (otherwise it should similarly neutralize the
inhibitor). This could involve the lipid indirectly affecting the KID
by modifying the local conformation. With Cdc42 activation, we
suggested that p21·GTP binding drives such a conformational switch in
the (overlapping) autoinhibitor region (7). This is borne out by
comparison of the recent structures of Cdc42·GTP complexed to PAK
CRIB/KID (28) with the PAK CRIB/KID complexed to the kinase domain (9).
Thus it is conceivable that sphingosine essentially mimics p21 binding by acting within the CRIB domain. To test this we first compared autophosphorylation mutants of
PAK for in vitro
activation induced by either sphingosine or Cdc42·GTP
S. As in
previous observations (Fig. 3)
PAKS144A/S149A and
PAKT422A proteins were activated to a lesser extent
in vitro by Cdc42·GTP
S (Fig. 6B, left
panel) than wild type kinase. By contrast sphingosine activation
in vitro, was significantly affected only by substitution at
Thr-422. Thus mutations of S144A/S149A affect kinase activation by
Cdc42·GTP but not by sphingosine. Differences in (tryptic) autophosphorylation profiles with the two activators (26) could reflect
masking of N-terminal sites (perhaps Ser-144 and Ser-149) by
sphingosine or preferential inhibition of the kinase toward some
autophosphorylation sites.

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Fig. 6.
Comparison of Cdc42 and sphingosine-mediated
activation. PAK and various substitution mutants as indicated
(100 ng) were assayed with or without Cdc42·GTP S (250 ng) or
sphingosine (100 µM) for phosphorylation of MBP. The
S144A/S149A mutant exhibited the same activity (within experimental
error) as wild type PAK in the presence of sphingosine.
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Fig. 7A shows how the extent
of
PAK autophosphorylation appears to differ during activation with
either Cdc42·GTP
S or soluble sphingosine. Under these conditions
the two activators show similar activity kinetics, yet sphingosine
autophosphorylation was apparently far from complete. An additive
effect of sphingosine and GTP
S.Cdc42 was seen in terms of PAK
activity (i.e. MBP phosphorylation). Activation using
sphingosine micelles (100 µM) with excess Cdc42·GTP
S under conditions where sphingosine was an ineffective activator demonstrated a synergistic effect on PAK activity (Fig. 6C).
Thus these two positive regulators may act at distinct sites and
potentially act in concert to stimulate
PAK in vivo. If
sphingosine interacts at a different site to Cdc42, substitutions in
the Cdc42 binding domain should not affect sphingosine-mediated
activation. Indeed
PAKI75N and
PAKS76P
mutants (7), which cannot bind GTP
S.Cdc42 were activated normally by
sphingosine (Fig. 8, A and
B).

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Fig. 7.
Cdc42 and sphingosine can cooperate in the
activation of PAK. A, purified PAK was incubated
with Cdc42·GTP S or 100 µM sphingosine at 32 °C in
the presence of 20% Me2SO (see B). At the time
shown aliquots of the assay mix were removed and subjected to Western
analysis with anti- PAK antibodies. B, the reaction
conditions were as above with the following assay conditions: 100 ng of
PAK, 250 ng of Cdc42·GTP S, and 5 µg of myelin basic protein
substrate per 50 µl of reaction volume. At the time shown aliquots
were withdrawn and added to 2× SDS sample buffer to quench the
reaction. Counts associated with MBP were quantified (shown below).
C, assay conditions were as above with the exception that
sonicated sphingosine vesicles were used.
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Fig. 8.
Cdc42-binding defective
PAK mutants are activated by sphingosine.
A, HA-tagged wild type PAK (WT) or the
substitution mutants I75N or S76P were purified using anti-HA
antibodies and incubated with Cdc42·GTP S and MBP under standard
conditions. Only the WT kinase was activated by this treatment as
indicated by the shift in mobility (top panel).
B, in the presence of 100 µM sphingosine both
mutants were activated similarly as assessed by MBP
phosphorylation.
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DISCUSSION |
PAK Autophosphorylation and Regulation of Kinase
Function--
Because PAK is autophosphorylated at several sites, it
is pertinent to ask what specific roles these might play. Here we have shown with both
- and
PAK that two conserved sites are key to regulating activity. The
PAK Thr-422 (within the catalytic domain) and a Ser-144 flanking the kinase inhibitor region play distinct but
complementary roles. Substitution of the autophosphorylated Thr-422 by
aspartic acid can render the
PAK kinase constitutively active
in vivo but not fully active by in vitro criteria
(11). However, substitution of the N-terminal autophosphorylation sites with acidic residues leads to proteins that are unstable in
vivo (data not shown).
There have been several previous investigations of differential PAK
autophosphorylation under various activation conditions. An analysis
using
PAK concluded that there were significant differences between
autophosphorylation with Mg·ATP alone compared with events occurring
in the presence of Cdc42 (24). Interestingly, it was observed that
Cdc42·GTP
S drives specific phosphorylation of Ser-141, and
Ser-165, and a higher levels of Thr-402 phosphorylation. This
PAK
Ser-141 (analogous to
PAK Ser-144) forms part of the kinase inhibitory domain (KID) that appears to be accessible only when Cdc42·GTP is bound. Upon phosphorylation this then prevents the KID
packing against the kinase domain (Fig.
9A). When Thr-422 is
subsequently transphosphorylated, this in turn prevents catalytic domain interaction with the KID (illustrated in Fig. 9B).
These multiple events may be designed to ensure that the kinase
switches from inactive to active state only under specific
conditions.

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Fig. 9.
A model for the events occurring during PAK
activation. A, based on the published structure
of Lei et al., composed of PAK1 complexed to a portion of
the N-terminal domain (9), the relevant backbone regions of the KID,
and kinase domains are shown where phosphorylation of serine 144 in the
PAK KID is predicted to cause steric hindrance with Arg-388 in the
catalytic domain. B, the interaction of Cdc42·GTP or
sphingosine with PAK leads to a conformational change in the KID
resulting in the dis-inhibition of the catalytic domain and
autophosphorylation in the regulatory region. Sphingosine leads to
phosphorylation of only a subset of sites (26) and probably binds to
and inhibits the kinase domain. In this proposed mechanism of
activation, phosphorylation of the activation loop threonine follows
N-terminal autophosphorylation. The trans-phosphorylation event is
facilitated by PAK dimerization (9), which is not illustrated directly
in the model.
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The autophosphorylation of the regulatory threonine, Thr-402
PAK
parallels activity of the kinase, suggesting that this threonine autophosphorylation event is closely coupled to the activation process
(29) as we have found here with
PAK. Equivalent threonine residues
are present in a motif that is conserved in all the PAK family kinases,
and corresponding peptides function as efficient substrates for PAK
(30). Recent experiments with
PAK have demonstrated a peculiar
difference between autophosphorylation of the N-terminal serine
residues and the single-threonine residue (18). When Mn·ATP is used
instead of Mg·ATP, the threonine is refractory to
autophosphorylation: Similarly, PAK substrates cannot be phosphorylated by Mn·ATP, substantiating the idea that the activation loop
requires intermolecular phosphorylation.
The N-terminal regulatory domain of PAK clearly functions to repress
catalytic activity of PAK: Deletion of sequences N-terminal to the
kinase domain by proteolysis renders PAK constitutively active (29).
Indeed PAK2 (
PAK) can undergo caspase-catalyzed proteolysis which
"releases" an active catalytic domain that appears responsible for
a number of morphological changes associated with apoptotic cells (10).
The issue that has not been fully addressed is the nature of the
activation step upon proteolysis. Because the N-terminal domain can
inhibit with high potency (Ki ~ 90 nM), and the catalytic domain can be co-crystallized with the inhibitory domain (9), proteolysis would not be expected to
actually release the catalytic domain. It is therefore likely that proteolysis of the holoenzyme actually drives a conformational change in the KID.
What is the purpose of having a two-step activation process? We
envision binding of Cdc42·GTP or Rac·GTP to structurally modify the
inhibitory domain allowing first autophosphorylation of the
PAK N
terminus, including Ser-144 (or equivalent residues for other
isoforms). Because
PAK exists as a dimer (9) Thr-422 transphosphorylation should be relatively efficient (Fig.
9B). If Nck and PIX are bound (16, 31), it is likely that
phosphorylation of the sites Ser-21, Ser-198, and Ser-203 would be
impaired. However, even following dissociation of the GTPase, the
kinase would remain in an "open" state allowing the intermolecular
activation of PAK by phosphorylation of Thr-422 to occur.
Alternatively, other kinases, including PDK1, might carry out
phosphorylation of the activation loop (32).
Mechanisms Underlying PAK Activation in Vivo--
It is already
established that integrin-dependent cell-matrix
interactions promote activation of Rac1 and Cdc42 and thus PAK (33,
34). The kinase in turn is coupled to PIX and PI3K (17). However, data
showing that integrin-mediated but not growth factor-mediated Rac·GTP
is responsible for activation of PAK (34) suggest that mechanisms exist
to segregate p21 targets from their partner GTPases. Whether the
differential localization of Rac1·GTP (rather than PAK itself) is the
key event that determines PAK activation remains to be established. It
seems unlikely that PAK localization is determined solely by its
partner GTPases. For example PAK activation within FCs will be limited
by the availability of binding sites with PIX·GIT1, which are
FC-localized via paxillin (35). In mammals PAKs can function within
FCs, but what of other organisms? We have previously shown that DPAK is
enriched in phosphotyrosine-rich structures at the leading edge of
Drosophila epithelial cells (5). In flies DPAK is implicated
in events downstream of Dock (the Nck homologue) during axonal
outgrowth (36), and circumstantial evidence suggests that the Rac1
activator in this system is Trio (37). Further genetic studies of
Drosophila PIX and GIT will no doubt bring us to a better
understanding of the signaling roles of these PAK partners.
Nck SH3 binding to PAK via the consensus motif
(PXXPXRXXS), is blocked by serine
autophosphorylation in this motif (25). Similarly, the PIX SH3 binding
site in PAKs (conserved in worms and flies) is flanked by two sites
whose autophosphorylation (Fig. 1A) blocks SH3 interaction
(25). When the PAK inhibitor
PAK-(83-149) is introduced into cells,
endogenous
PAK becomes stably associated with the
PIX·GIT1·paxillin complex in FCs. The dynamic nature of the
interaction thus depends on the two sites that control
PAK activity
(Ser-144 and Thr-422) and the three sites that regulate association
with SH3 containing partners (Ser-21, Ser-198, and Ser-203). PAK is
implicated in promoting cell migration (14, 38): We envision this as a
multistep process involving the activation of Rac1 (via PIX) coupled to
the turnover of existing focal complexes via GIT1 (35). The generation
of new peripheral focal complexes is driven by the Cdc42 effector
myotonin related Cdc42-binding kinase (39). The molecular
mechanism of PAK activation by sphingosine is distinct from that
induced by Cdc42 (12) as also demonstrated here. At present it is not
clear how important a role sphingosine plays in PAK regulation in
vivo, and its possible connection to cell motility warrants
further investigation.