 |
INTRODUCTION |
Three distinct isoforms of
PAK1 are known to be
expressed in mammalian cells: the brain-enriched
-PAK (1) also
termed PAK1 (2), the rat
-PAK isoform (3) also known as mouse PAK3 (4), and the ubiquitous
-PAK (5, 6) also referred to as PAK2 (7) or
PAK I (8). These PAKs are characterized by a highly conserved domain in
the N-terminal, regulatory region for binding of small G-proteins such
as Cdc42 and Rac. This interaction stimulates autophosphorylation and
activation of PAK (9, 10).
Historically,
-PAK was first identified in rabbit reticulocytes as
an inactive holoenzyme, whose protein kinase activity toward substrates
such as histone 2B and histone 4 was inducible by limited tryptic
digestion (11, 12). Over the past few years, PAK activation has been
implicated in a number of signaling pathways, including those
stimulated during cell growth and differentiation (13-15), stress
(16), and apoptosis (17-19). However, within a broad range of
biochemical contexts, autophosphorylation has been consistently
reported as a common trait of
-PAK activation.
Recently
-PAK has been shown to be activated by sphingolipids (20),
while some substrates (e.g. histones 4 and 2B) were found to
induce activation of
-PAK (21). The finding that
-PAK undergoes
autophosphorylation on residues in the N-terminal region and on a
single residue in the C-terminal domain (22) is consistent with a role
for the N terminus in regulating the protein kinase activity of PAK.
Previous studies from this laboratory (17) have shown that
autophosphorylation of the regulatory and catalytic domains is required
for activation of
-PAK following caspase cleavage. Taken together,
these data suggest that multisite autophosphorylation regulates the
activity of this family of kinases. However, to date, only the
autophosphorylation sites on
-PAK have been fully described (22).
The only autophosphorylation site identified in
-PAK is Thr-402,
which has been shown to be involved in activation of the protein kinase
(17, 23, 24).
One of the main objectives of this study is to understand which of the
multiple autophosphorylation events is involved in
-PAK activation.
Taking into consideration the possibility that the post-translational
changes of a baculovirus-expressed form of
-PAK might be
substantially different from those occurring in mammalian cells,
two-dimensional profiles of recombinant
-PAK were compared with
native
-PAK purified from rabbit reticulocytes, before and after
exposure to Cdc42(GTP
S) or histone II-AS. Tryptic phosphopeptides
were generated by digestion of the autophosphorylated recombinant
-PAK and fractionated using a two-dimensional peptide gel system
(25). Comparative phosphopeptide mapping was carried out to
characterize the phosphorylation status of inactive and active
-PAK
and to allow the identification of those phosphopeptides whose presence
is indicative of
-PAK activation. The sequence of each
phosphopeptide was identified by automated sequencing, while the
position of the phosphoresidue was determined by monitoring the release
of radioactivity via manual Edman degradation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
All chemicals were from Sigma if not otherwise
indicated. Ampholines (pH 3.5-10 and 3.5-5) were from Amersham
Pharmacia Biotech; Zwittergent 3-16 was from Calbiochem;
nitrocellulose was from Schleicher & Schuell. The polyclonal antibody
to
-PAK protein was prepared in rat as described previously (26).
Anti-Ste20-subdomain VI antibody (anti-Ste20-VI) that is specific for
the catalytic domain of all three isoforms of PAK was purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). Horseradish
peroxidase-conjugated secondary antibodies were from Organon Teknika.
The chemioluminescent detection system was purchased from Amersham
Pharmacia Biotech. Cellophane membrane backing sheets were from
Bio-Rad, C18 cartridges (Sep-Pak, 50 mg) were from Waters, and
arylamine disks (Sequelon-AA type) were from PerSeptive
Biosystems. A sequencer (Procise 492) from Applied Biosystems
was used for amino acid microsequencing. The peptide substrate S3
(AKRESAA) was synthesized as described previously (26).
Preparation of
-PAK--
The cDNA for rabbit
-PAK was
cloned into the pAcG2T vector and used to transfect TN5B-4 cells as
described elsewhere (17). The recombinant protein was purified by
affinity chromatography on glutathione-Sepharose, cleaved with thrombin
and eluted (17). After thrombin cleavage, the N terminus of the
recombinant protein contained the following five residues derived from
the fusion protein: Gly, Ser, Leu, Gly, and His. Native
-PAK was
purified from the postribosomal supernatant of rabbit reticulocytes by chromatography on DEAE-cellulose, SP-Sepharose, protamine-agarose, and
Mono Q columns (24).
In Vitro Phosphorylation Assays--
Autophosphorylation of
native (0.05-0.1 µg) and recombinant (0.5-5 µg)
-PAK was
carried out in a 20-40 µl reaction mixture containing 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2,
30 mM
-mercaptoethanol, and 0.2 mM
[
-32P]ATP (1000 cpm/pmol). Cdc42 (0.5-2 µg),
prepared as described by Jakobi and colleagues (8), was preloaded with
0.18 mM GTP
S for 10 min at 30 °C prior to addition to
the reaction. When indicated, histone II-AS was added at a final
concentration of 1 mg/ml. The autophosphorylation reactions were
terminated after 30 min at 30 °C by the addition of either 20 µl
of 2× concentrated sample buffer for standard polyacrylamide gel
electrophoresis (SDS-PAGE) or 100 µl of sample buffer for
two-dimensional gel electrophoresis (two-dimensional PAGE).
For the analysis of substrate phosphorylation, the above reaction
contained peptide S3 (1 mM), a synthetic substrate of
-PAK (27). After incubation, the reaction mix was diluted with 6 M urea in 125 mM Tris-HCl, pH 6.8, and
subjected to a 40% alkaline peptide PAGE, as described by West and
colleagues (28). On the basis of the high reticulation feature, this
gel allowed an accurate resolution of the peptide band and the
subsequent quantitation of phosphorylation.
Gel Electrophoresis and Immunorecognition--
Samples were
either subjected to 10% SDS-PAGE (29) or to two-dimensional PAGE,
consisting of gel isoelectrofocusing followed by 10% SDS-PAGE as
described previously (30). Proteins were electrotransferred onto a
nitrocellulose membrane (31) and stained with 0.5% (w/v) Ponceau in
1% acetic acid prior to autoradiography. When indicated, the proteins
were stained with 0.1% (w/v) Amido Black in 25% methanol and 10%
acetic acid.
For Western blotting, the nitrocellulose was probed with a polyclonal
anti-PAK antibody, as described previously (26). This antibody has been
shown to recognize
-PAK (26) and to react specifically with the
regulatory region (17). In some cases, immunorecognition was carried
out with anti-Ste20-VI, a commercially available antibody raised
against a peptide of the catalytic domain, which immunoreacted with
-,
-, and
-PAK. Immunoreactivity was detected with
peroxidase-conjugated secondary antibody using the ECL method.
Peptide Gel Electrophoresis--
After one- or two-dimensional
electrophoresis and transfer, 32P-labeled
-PAK was
excised from the nitrocellulose and digested with trypsin, as described
(32). 32P-Labeled tryptic phosphopeptides were analyzed by
two-dimensional peptide PAGE, as described elsewhere (25). Briefly,
aliquots (20-80 µl) of the digested protein were subjected to
nondenaturing isoelectric focusing in gel tubes followed in the second
dimension by electrophoresis on a 40% polyacrylamide alkaline slab
gel. Prior to autoradiography, the gel was coated with a cellophane membrane backing sheet and dried under vacuum. When indicated, the
samples extracted from individual labeled spots of two-dimensional peptide gels were subjected to 16% SDS-PAGE in Tris-Tricine buffer, as
described by Schagger and von Jagow (33).
Extraction and Cleanup of Selected Phosphopeptides--
After
autoradiography, the radioactive tryptic phosphopeptides were
individually excised from the 40% gel and incubated in 2 ml of 0.1%
trifluoroacetic acid overnight at room temperature. The eluted
phosphopeptides were subjected to a cleanup procedure using C18
cartridges (50 mg), as described elsewhere (25). The final eluates were
taken to dryness prior to Cerenkov counting of the radioactivity.
Manual and Automated Sequencing--
Each lyophilized
phosphopeptide was dissolved in 20 µl of 30% acetonitrile and 0.1%
trifluoroacetic acid and covalently bound to a disk of arylamine
membrane (Sequelon-AA type) as described by the supplier. The disk was
routinely cut in two parts: approximately 3/4 of the disk was
used for automated sequencing, while the remaining 1/4 of the
disk was subjected to manual Edman degradation as described by Sullivan
and Wong (34).
 |
RESULTS |
Detection of Differentially Phosphorylated Forms of
-PAK--
Multiple autophosphorylation of
-PAK was analyzed by
two-dimensional PAGE. In order to visualize the effect of
autophosphorylation, both native
-PAK purified from rabbit
reticul- ocytes and recombinant
-PAK expressed in
baculovirus-infected insect cells were incubated with Cdc42(GTP
S) in
the presence of [
-32P]MgATP. After two-dimensional
PAGE, the proteins were transferred onto nitrocellulose and detected
with an antibody previously shown to recognize the regulatory domain of
-PAK (17). Using this approach,
-PAK was resolved into a number
of differentially migrating immunoreactive species with distinct pI
values (Fig. 1A). Treatment of
native
-PAK with activated Cdc42 produced an acidic shift in the
immunoreactive profile of
-PAK (Fig. 1B), with a
disappearance of the forms migrating 4.8-5.5 cm from the basic origin
and the concomitant appearance of
-PAK forms migrating further
(5.4-6.2 cm). To verify the correspondence between changes in the
electrophoretic profile and autophosphorylation, Cdc42-stimulated,
32P-labeled
-PAK was also detected by autoradiography.
The vast majority of the radiolabeled native
-PAK was present in the
highly acidic forms that migrated 5.4-6.2 cm from the basic origin,
while little radioactivity was associated with the other isoelectric forms. (Fig. 1C).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 1.
Two-dimensional analysis of native
-PAK before and after Cdc42-stimulated
autophosphorylation. In vitro autophosphorylation of native
-PAK was carried out in the absence (A) or in the
presence (B and C) of [ -32P]ATP
and Cdc42(GTP S). Native -PAK was subjected to two-dimensional
electrophoresis prior to transfer onto a nitrocellulose membrane.
A and B, autoradiograms of immunoblots with
anti-PAK antibody that specifically recognizes the regulatory domain of
-PAK. C, autoradiogram of 32P-labeled -PAK
using the nitrocellulose membrane shown in B. The ruler
facilitates visualization of the
autophosphorylation-dependent mobility shift by measuring
the distance from the basic origin of the first dimensional gel.
Approximate molecular mass is indicated on the right,
according to the migration of markers such as bovine serum albumin (68 kDa), actin (45 kDa), and soybean trypsin inhibitor (20 kDa). 1st
D, first dimension; 2nd D, second dimension.
|
|
When recombinant
-PAK was subjected to the same experimental
protocol, the resulting immunoreactivity and autoradiographic profiles
were similar, if not identical, to those of native
-PAK. The acidic
change in the profile after Cdc42-stimulated autophosphorylation resulted in the disappearance of forms migrating 4.8-5.5 cm from the
basic origin (Fig. 2A), with
the concomitant appearance of
-PAK forms migrating at 5.5-6.7 cm
(Fig. 2B). The latter corresponded to the highly
acidic radiolabeled forms as assessed by autoradiography (Fig.
2C).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 2.
Two-dimensional analysis of recombinant
-PAK before and after Cdc42-stimulated
autophosphorylation. This figure is similar to Fig. 1,
except that recombinant -PAK was examined.
|
|
When recombinant
-PAK was incubated with MgATP only, an acidic shift
was detected via protein staining and immunorecognition (Fig.
3), indicating an increase in phosphate
over the mock-treated sample. However, this shift was limited as
compared with that obtained in the presence of Cdc42(GTP
S) (compare
Fig. 3D with Fig. 2B). This supports the
conclusion that Cdc42(GTP
S) is required for maximal
autophosphorylation of
-PAK. Analogous results were obtained when
parallel two-dimensional blots were probed with anti-Ste20-VI (data not
shown).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 3.
Autophosphorylation of recombinant
-PAK in the absence of
Cdc42(GTP S). In vitro
autophosphorylation of recombinant -PAK was carried out in the
absence (A and C) or in the presence
(B and D) of MgATP. After two-dimensional
electrophoresis and transfer onto nitrocellulose membranes, -PAK was
immunodetected with anti-PAK antibody or stained with Amido Black.
1st D, first dimension; 2nd D, second
dimension.
|
|
Phosphopeptide Mapping--
Having found substantial identity in
the two-dimensional immunoblot profiles of native and recombinant
-PAK, we set out to investigate how changes in
-PAK
autophosphorylation correlated with protein kinase activity toward
exogenous substrates, and in particular whether specific
phosphopeptides were indicative of
-PAK activation. Following
incubation of
-PAK with radiolabeled MgATP alone and in the presence
of the activators Cdc42(GTP
S) or histone II-AS,
-PAK
autophosphorylation was assessed by autoradiography following SDS-PAGE.
Jakobi and colleagues (8, 21) have shown that Cdc42(GTP
S) or histone
4 stimulated autophosphorylation of recombinant
-PAK up to 4-fold.
In the present study,
-PAK autophosphorylation was stimulated 5.1- and 6.2-fold by Cdc42(GTP
S) and histone II-AS, respectively
(Fig. 4).

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
Comparative phosphopeptide mapping of active
and inactive -PAK. Baculovirus-expressed
-PAK was autophosphorylated in vitro with MgATP alone
(A), MgATP plus Cdc42(GTP S) (B), or MgATP plus
histone II-AS (C). Aliquots of the respective tryptic
digests, consisting of 7500, 13,000, and 10,000 cpm, were subjected to
two-dimensional peptide gel electrophoresis as shown in
A-C. 1st D, first dimension; 2nd D,
second dimension.
|
|
After tryptic digestion of autophosphorylated
-PAK, two-dimensional
phosphopeptide maps were obtained by a combination of native
isoelectrofocusing and high reticulation polyacrylamide peptide gels
(25). The radiolabeled spots with assigned numbers, as shown in Fig. 4,
represent the major phosphopeptides of
-PAK that were consistently
observed in repeated experiments. A qualitatively similar profile was
obtained with in vitro autophosphorylated native
-PAK
(data not shown). Comparative phosphopeptide mapping showed a clear
difference between the phosphopeptide maps of
-PAK incubated only
with MgATP (Fig. 4A) and
-PAK activated by Cdc42 (Fig.
4B). Consistent with the observation that histone was
capable of activating
-PAK (21), the phosphopeptide map of
-PAK
autophosphorylated in the presence of histone II-AS (Fig.
4C) was identical to the map of Cdc42-activated
-PAK.
Upon repeated experiments, the specific accumulation of spots 7, 9, 11, and 13 in the samples of
-PAK treated with Cdc42 or histone
suggested a correlation between the acquisition of a specific
phosphorylation state and protein kinase activation.
To show that
-PAK was activated by Cdc42,
-PAK autophosphorylated
in the presence or absence of Cdc42(GTP
S) was assayed with peptide
S3. Autoradiography of the dried gel (Fig.
5) following alkaline 40% PAGE showed a
3-fold increase in
-PAK activity in the presence of Cdc42(GTP
S).
The activity of
-PAK in the absence of Cdc42 was probably due to
small amounts of fully autophosphorylated and activated forms in the
recombinant
-PAK sample, as suggested by the presence of trace
amounts of spots 7, 9, and 13 in the respective two-dimensional peptide
map (see Fig. 4).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 5.
Protein kinase activity of
-PAK assayed with peptide S3. Recombinant
-PAK was assayed in the presence and absence of Cdc42(GTP S) with
S3, a synthetic peptide containing a site for -PAK (26). After
electrophoresis on a 40% alkaline peptide gel as described under
"Experimental Procedures," 32P-incorporated into S3 was
quantitated by Cerenkov counting of the excised gel bands.
|
|
Identification of Autophosphorylation Sites--
To identify the
sequence and the phosphoresidue of the major phosphopeptides from
Cdc42-stimulated
-PAK, an experimental strategy based on the
combination of automated and manual amino acid sequencing was employed
as described elsewhere (25). In particular, the major radiolabeled
spots 4, 9, 10, 11, 12, and 13 from the two-dimensional peptide gel of
Cdc42-stimulated
-PAK were isolated and subjected to a cleanup
procedure prior to sequence analysis. The amount of phosphopeptide
present in the minor spots was too small to be successfully sequenced
(data not shown).
In case of spots 10, 12, and 13, the amino acids were unambiguously
identified by automated sequencing, and the vast majority of the
32P was released at a single cycle during manual
sequencing. The information resulting from manual Edman degradation and
from automated sequencing clearly identified Ser-192, Ser-197, and
Thr-402 as the phosphorylation sites of phosphopeptides present in
spots 10, 12, and 13, respectively (Fig.
6, A-C).

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 6.
Sequencing of spots 4, 10, 12, and 13 from -PAK autophosphorylated in the presence
of Cdc42(GTP S). A, spot 10;
B, spot 12; C, spot 13; D, spot 4. The
manual and automated sequencing data resulting from Cdc42-stimulated
autophosphorylation of -PAK are shown. The cycle at which
32P was released by manual Edman degradation and the
identified amino acid sequence are indicated together with the sequence
of the respective peptide as derived from the cDNA (8).
X designates amino acid residues that could not be
identified by automated sequencing. The underlined residues
are those found to be phosphorylated. 1st D, first
dimension; 2nd D, second dimension.
|
|
In case of phosphopeptide 4, manual sequencing revealed a substantial
release of 32P at both cycles 2 and 3 (Fig. 6D).
Both of these residues were serine, rendering it impossible to deduce
whether the peptide was diphosphorylated or consisted of a mixture of
two phosphopeptides, each one phosphorylated on a single site. For a
number of reasons explained under "Discussion," we conclude that
the latter option is the most plausible.
Another case in which the majority of radioactivity was not released at
a single cycle of Edman degradation was spot 9 (Fig. 7A). As shown by automated
sequencing, this spot consisted of a peptide starting with Val-161,
which was phosphorylated at Ser-165, plus a second peptide, which
started with phosphorylated Ser-197. The presence of the latter, a
proteolytic product of phosphopeptide 12, explained why radioactivity
was released in the first as well as the fourth cycle. That spot 9 consisted of more than a single phosphopeptide was confirmed by close
inspection of a two-dimensional peptide map (Fig. 7B),
wherein the time of isoelectrofocusing in the first dimension was
extended to maximize the resolution of phosphopeptides with different
pIs.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 7.
Analysis of spot 9 from
-PAK autophosphorylated in the presence of
Cdc42(GTP S). A, summary of manual
and automated sequencing data resulting from analysis of
Cdc42-stimulated autophosphorylation of -PAK. The presence of two
distinct peptides was deduced by taking into consideration the first
(1st) as well as the second (2nd) sequence
identified by the sequencer. B, the autoradiogram of spot 9 from a two-dimensional peptide gel, wherein the first dimensional
isoelectrofocusing was run until the tracking dye reached the end of
the gel.
|
|
Similarly, in case of spot 11, automated sequencing revealed the
presence of two equally abundant tryptic phosphopeptides, namely the
peptide starting with Ile-53, which was phosphorylated on Ser-55, plus
the peptide starting from Tyr-139 and phosphorylated on Ser-141 (Fig.
8A). These two peptides not
only comigrated in the two-dimensional peptide gel system but also had
in common a phosphoserine in the third position. However, when
autophosphorylation of
-PAK was carried out in the absence of
Cdc42(GTP
S), spot 11 contained a predominant phosphopeptide starting
with Ile-53 and trace amounts of a second phosphopeptide, as shown by
automated sequencing (Fig. 8B). The notion that spot 11 of
inactive
-PAK consisted of one major phosphopeptide, while two
distinct phosphopeptides were present equally in spot 11 of
Cdc42-stimulated
-PAK, was confirmed by submitting the samples to
another electrophoretic system consisting of a 16% polyacrylamide gel
run in Tris-Tricine buffer containing SDS (Fig. 8C). The
presence of SDS in the latter gel allowed the resolution of these
phosphopeptides. This conclusion is fully consistent with previous
sequence analysis (25) of autophosphorylation sites in the N-terminal
peptide of
-PAK. Therefore, comparative phosphopeptide mapping
allowed a clear identification of Ser-55 and Ser-141 as two distinct
sites in spot 11 from Cdc42-activated
-PAK, and of Ser-55 as the
only site in spot 11 after autophosphorylation of
-PAK with MgATP alone.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 8.
Analysis of spot 11 from
-PAK autophosphorylated in the presence or absence
of Cdc42(GTP S). A and B,
summary of manual and automated sequencing data resulting from -PAK
autophosphorylation in the presence (+) and absence ( ) of
Cdc42(GTP S), respectively. C, analysis of the sample
isolated from spot 11 in A via 16% SDS-PAGE in Tris-Tricine
buffer, as described by Schagger and von Jagow (33).
|
|
 |
DISCUSSION |
Several baculovirus-expressed mammalian proteins have been
reported to be functionally indistinguishable from the corresponding native proteins. It has also been observed that the sites
phosphorylated on proteins expressed in baculovirus-infected insect
cells and mammalian cells are often the same (35, 36). In the present study, we compared the immunoblot profiles of native and recombinant
-PAK by two-dimensional PAGE. Both recombinant and native forms were
resolved into several differentially migrating forms, thus validating
the choice to use the baculovirus-expressed
-PAK for sequencing the
autophosphorylation sites.
Comparative phosphopeptide mapping of
-PAK autophosphorylation with
or without Cdc42(GTP
S) or histone II-AS revealed that spots 7, 9, and 13 were present only in the map of activated
-PAK and that spot
11 was increased (see Fig. 4). As aforementioned, recent evidence has
indicated that autophosphorylation is a common feature of
-PAK
activation within a broad variety of signaling pathways. Therefore, the
autophosphorylation pattern, as assessed by two-dimensional
phosphopeptide mapping, may represent a useful index of PAK activation.
This is supported by our previous finding that
-PAK
autophosphorylation precedes activation, regardless of whether the
holoenzyme is activated by Cdc42(GTP
S) or by caspase 3 (17, 24).
To directly investigate the exact positions of the Cdc42-inducible
autophosphorylation sites, we employed a combination of high resolution
phosphopeptide mapping with manual and automated amino acid sequencing
(25). In most cases, each spot on the two-dimensional peptide map
consisted of an individual phosphopeptide whose radioactivity was
released primarily during a single cycle, indicating that the majority
of phosphopeptides contained a single autophosphorylation site. Manual
sequencing of spot 4 revealed an approximately equal release of
radioactivity in the second and third cycle, corresponding to Ser-19
and Ser-20, respectively. There are at least three potential
explanations for such an outcome: (i) artifactual tailing of
32P release at the cycle corresponding to the residue
adjacent to the bona fide phosphoresidue; (ii) dual phosphorylation at
the adjacent sites of Ser-19 and Ser-20; and (iii) partial
phosphorylation at either Ser-19 or Ser-20. For a number of reasons,
the first two options do not apply to this case. First, in our hands
the extent of 32P tailing deriving from a site adjacent to
a phosphoresidue is routinely very limited. Second, in line with the
typically extensive degradation of the phenylthiohydantoin-derivative
of phosphoserine (37), dual phosphorylation of Ser-19 and Ser-20 would
be expected to generate little if any of the corresponding
phenylthiohydantoin-derivative. This was not the case;
phenylthiohydantoin-derivatives of Ser-19 and Ser-20 were clearly
detected upon automated sequencing. Such an observation suggests the
existence of two distinct pools of
-PAK phosphorylated on either
Ser-19 or Ser-20. Alternative phosphorylation on adjacent serine
residues resulting in a pair of functionally undistinguishable
phosphoprotein species has already been reported in the literature
(38).
A summary of the sequencing data is presented in Table
I. Autophosphorylation of
-PAK with
MgATP alone takes place at Ser-19, Ser-20, Ser-55, Ser-192, and
Ser-197. In the presence of Cdc42, additional autophosphorylation of
-PAK occurs at Ser-141, Ser-165, and Thr-402. Thus, the latter sites
are selectively phosphorylated upon
-PAK activation. In
-PAK, the
phosphorylation of Thr-422, which corresponds to Thr-402 of
-PAK,
has been shown to be critical for the catalytic function of the protein
kinase (22).
View this table:
[in this window]
[in a new window]
|
Table I
Summary of data from manual and automated sequencing of
autophosphorylated -PAK
The cycle at which 32P was released upon manual Edman
degradation, the sequence of the respective phosphopeptide, and the
phosphorylated residue for each of the assigned spots are indicated
with respect to -PAK autophosphorylation in the presence or absence
of Cdc42(GTP S). The various lengths of the examined sequences are
due to differential requirements for unambiguous identification of the
phosphopeptides.
|
|