Multisite Autophosphorylation of p21-activated Protein Kinase gamma -PAK as a Function of Activation*

Andrea Gatti, Zhongdong Huang, Polygena T. Tuazon, and Jolinda A. TraughDagger

From the Department of Biochemistry, University of California, Riverside, California 92521

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous gamma -isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma -PAK, while native gamma -PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of gamma -PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma -PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma -PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, gamma -PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma -PAK, six of which are in common with those previously reported in alpha -PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma -form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma -PAK activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Three distinct isoforms of PAK1 are known to be expressed in mammalian cells: the brain-enriched alpha -PAK (1) also termed PAK1 (2), the rat beta -PAK isoform (3) also known as mouse PAK3 (4), and the ubiquitous gamma -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, gamma -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 gamma -PAK activation.

Recently alpha -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 gamma -PAK (21). The finding that alpha -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 gamma -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 alpha -PAK have been fully described (22). The only autophosphorylation site identified in gamma -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 gamma -PAK activation. Taking into consideration the possibility that the post-translational changes of a baculovirus-expressed form of gamma -PAK might be substantially different from those occurring in mammalian cells, two-dimensional profiles of recombinant gamma -PAK were compared with native gamma -PAK purified from rabbit reticulocytes, before and after exposure to Cdc42(GTPgamma S) or histone II-AS. Tryptic phosphopeptides were generated by digestion of the autophosphorylated recombinant gamma -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 gamma -PAK and to allow the identification of those phosphopeptides whose presence is indicative of gamma -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 gamma -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 gamma -PAK-- The cDNA for rabbit gamma -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 gamma -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) gamma -PAK was carried out in a 20-40 µl reaction mixture containing 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 30 mM beta -mercaptoethanol, and 0.2 mM [gamma -32P]ATP (1000 cpm/pmol). Cdc42 (0.5-2 µg), prepared as described by Jakobi and colleagues (8), was preloaded with 0.18 mM GTPgamma 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 gamma -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 gamma -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 alpha -, beta -, and gamma -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 gamma -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 gamma -PAK-- Multiple autophosphorylation of gamma -PAK was analyzed by two-dimensional PAGE. In order to visualize the effect of autophosphorylation, both native gamma -PAK purified from rabbit reticul- ocytes and recombinant gamma -PAK expressed in baculovirus-infected insect cells were incubated with Cdc42(GTPgamma S) in the presence of [gamma -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 gamma -PAK (17). Using this approach, gamma -PAK was resolved into a number of differentially migrating immunoreactive species with distinct pI values (Fig. 1A). Treatment of native gamma -PAK with activated Cdc42 produced an acidic shift in the immunoreactive profile of gamma -PAK (Fig. 1B), with a disappearance of the forms migrating 4.8-5.5 cm from the basic origin and the concomitant appearance of gamma -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 gamma -PAK was also detected by autoradiography. The vast majority of the radiolabeled native gamma -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).


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Fig. 1.   Two-dimensional analysis of native gamma -PAK before and after Cdc42-stimulated autophosphorylation. In vitro autophosphorylation of native gamma -PAK was carried out in the absence (A) or in the presence (B and C) of [gamma -32P]ATP and Cdc42(GTPgamma S). Native gamma -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 gamma -PAK. C, autoradiogram of 32P-labeled gamma -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 gamma -PAK was subjected to the same experimental protocol, the resulting immunoreactivity and autoradiographic profiles were similar, if not identical, to those of native gamma -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 gamma -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).


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Fig. 2.   Two-dimensional analysis of recombinant gamma -PAK before and after Cdc42-stimulated autophosphorylation. This figure is similar to Fig. 1, except that recombinant gamma -PAK was examined.

When recombinant gamma -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(GTPgamma S) (compare Fig. 3D with Fig. 2B). This supports the conclusion that Cdc42(GTPgamma S) is required for maximal autophosphorylation of gamma -PAK. Analogous results were obtained when parallel two-dimensional blots were probed with anti-Ste20-VI (data not shown).


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Fig. 3.   Autophosphorylation of recombinant gamma -PAK in the absence of Cdc42(GTPgamma S). In vitro autophosphorylation of recombinant gamma -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, gamma -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 gamma -PAK, we set out to investigate how changes in gamma -PAK autophosphorylation correlated with protein kinase activity toward exogenous substrates, and in particular whether specific phosphopeptides were indicative of gamma -PAK activation. Following incubation of gamma -PAK with radiolabeled MgATP alone and in the presence of the activators Cdc42(GTPgamma S) or histone II-AS, gamma -PAK autophosphorylation was assessed by autoradiography following SDS-PAGE. Jakobi and colleagues (8, 21) have shown that Cdc42(GTPgamma S) or histone 4 stimulated autophosphorylation of recombinant gamma -PAK up to 4-fold. In the present study, gamma -PAK autophosphorylation was stimulated 5.1- and 6.2-fold by Cdc42(GTPgamma S) and histone II-AS, respectively (Fig. 4).


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Fig. 4.   Comparative phosphopeptide mapping of active and inactive gamma -PAK. Baculovirus-expressed gamma -PAK was autophosphorylated in vitro with MgATP alone (A), MgATP plus Cdc42(GTPgamma 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 gamma -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 gamma -PAK that were consistently observed in repeated experiments. A qualitatively similar profile was obtained with in vitro autophosphorylated native gamma -PAK (data not shown). Comparative phosphopeptide mapping showed a clear difference between the phosphopeptide maps of gamma -PAK incubated only with MgATP (Fig. 4A) and gamma -PAK activated by Cdc42 (Fig. 4B). Consistent with the observation that histone was capable of activating gamma -PAK (21), the phosphopeptide map of gamma -PAK autophosphorylated in the presence of histone II-AS (Fig. 4C) was identical to the map of Cdc42-activated gamma -PAK. Upon repeated experiments, the specific accumulation of spots 7, 9, 11, and 13 in the samples of gamma -PAK treated with Cdc42 or histone suggested a correlation between the acquisition of a specific phosphorylation state and protein kinase activation.

To show that gamma -PAK was activated by Cdc42, gamma -PAK autophosphorylated in the presence or absence of Cdc42(GTPgamma S) was assayed with peptide S3. Autoradiography of the dried gel (Fig. 5) following alkaline 40% PAGE showed a 3-fold increase in gamma -PAK activity in the presence of Cdc42(GTPgamma S). The activity of gamma -PAK in the absence of Cdc42 was probably due to small amounts of fully autophosphorylated and activated forms in the recombinant gamma -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).


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Fig. 5.   Protein kinase activity of gamma -PAK assayed with peptide S3. Recombinant gamma -PAK was assayed in the presence and absence of Cdc42(GTPgamma S) with S3, a synthetic peptide containing a site for gamma -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 gamma -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 gamma -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).


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Fig. 6.   Sequencing of spots 4, 10, 12, and 13 from gamma -PAK autophosphorylated in the presence of Cdc42(GTPgamma 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 gamma -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.


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Fig. 7.   Analysis of spot 9 from gamma -PAK autophosphorylated in the presence of Cdc42(GTPgamma S). A, summary of manual and automated sequencing data resulting from analysis of Cdc42-stimulated autophosphorylation of gamma -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 gamma -PAK was carried out in the absence of Cdc42(GTPgamma 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 gamma -PAK consisted of one major phosphopeptide, while two distinct phosphopeptides were present equally in spot 11 of Cdc42-stimulated gamma -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 gamma -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 gamma -PAK, and of Ser-55 as the only site in spot 11 after autophosphorylation of gamma -PAK with MgATP alone.


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Fig. 8.   Analysis of spot 11 from gamma -PAK autophosphorylated in the presence or absence of Cdc42(GTPgamma S). A and B, summary of manual and automated sequencing data resulting from gamma -PAK autophosphorylation in the presence (+) and absence (-) of Cdc42(GTPgamma 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 gamma -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 gamma -PAK for sequencing the autophosphorylation sites.

Comparative phosphopeptide mapping of gamma -PAK autophosphorylation with or without Cdc42(GTPgamma S) or histone II-AS revealed that spots 7, 9, and 13 were present only in the map of activated gamma -PAK and that spot 11 was increased (see Fig. 4). As aforementioned, recent evidence has indicated that autophosphorylation is a common feature of gamma -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 gamma -PAK autophosphorylation precedes activation, regardless of whether the holoenzyme is activated by Cdc42(GTPgamma 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 gamma -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 gamma -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 gamma -PAK occurs at Ser-141, Ser-165, and Thr-402. Thus, the latter sites are selectively phosphorylated upon gamma -PAK activation. In alpha -PAK, the phosphorylation of Thr-422, which corresponds to Thr-402 of gamma -PAK, has been shown to be critical for the catalytic function of the protein kinase (22).

                              
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Table I
Summary of data from manual and automated sequencing of autophosphorylated gamma -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 gamma -PAK autophosphorylation in the presence or absence of Cdc42(GTPgamma S). The various lengths of the examined sequences are due to differential requirements for unambiguous identification of the phosphopeptides.

Due to incomplete tryptic digestion, not all of the 32P-labeled spots represent distinct phosphorylation sites. For example, phosphorylated Ser-197 is present both in spots 9 and 12 in the phosphopeptide map from Cdc42-stimulated gamma -PAK; the corresponding phosphopeptides have different sizes but share the same N terminus. Accumulation of the smaller phosphopepeptide starting with Ser-197 seems to be dependent on gamma -PAK autophosphorylation during activation, given that spot 9 is barely detectable with MgATP alone. In addition, Thr-402, besides being the single phosphorylation site in phosphopeptide 13, is also present in the minor spot 7, as indicated by manual sequencing and by phosphopeptide mapping of the C-terminal fragment resulting from cleavage of gamma -PAK by caspase 3 (data not shown).

The eight autophosphorylation sites identified in gamma -PAK from rabbit have similar positions in rat (5) and are thus likely to be autophosphorylated. However, only seven of the sites are present in human gamma -PAK, with Ser-165 replaced by proline (7). A comparison of the gamma -PAK autophosphorylation sites here identified in rabbit with those reported previously for alpha -PAK from rat shows that six of the sites are conserved (Fig. 9). In alpha -PAK, Ser-19 is replaced with threonine and has not been identified as a phosphoamino acid (22), while Ser-165 is replaced by proline. An additional phosphorylated serine is present in alpha -PAK as Ser-149; this site corresponds to glutamic acid at residue 146 in rabbit, rat, and human gamma -PAK (5-8), while the beta -PAK sequence from rat, mouse, and human contains aspartic acid (3, 4, 39). In beta -PAK, Ser-19 and Ser-165 are replaced by asparagine and proline, respectively.


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Fig. 9.   Sequence alignment and identification of autophosphorylation sites in gamma -PAK, as compared with alpha -PAK. The deduced amino acid sequences of rabbit gamma -PAK (8) and rat alpha -PAK (22) are aligned, and the phosphorylation sites are indicated (black squares).

Thus, Ser-165 is uniquely present in the rabbit and rat isoforms of gamma -PAK, while Ser-19 is unique to all forms of gamma -PAK. As phosphorylation of the corresponding Thr-20 of alpha -PAK has not been identified at this time (22), the possibility exists that such a site is exclusively phosphorylated in gamma -PAK. Since phosphorylation of Ser-141 is implicated in the activation of gamma -PAK and was detected in alpha -PAK, it is of interest that the phosphoserine 149 in alpha -PAK (corresponding to residue 146 in gamma -PAK) is already an acidic amino acid in the gamma  and beta  isoforms. This suggests that both of these residues may have an important role in PAK activation.

    ACKNOWLEDGEMENTS

We thank Dr. Rolf Jakobi and Barbara Walter for cloning, expression, and purification of recombinant gamma -PAK.

    FOOTNOTES

* This study was supported by U.S. Public Health Service Grant GM26738 and National Science Foundation Grant BIR-9601810.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Fax: 909-787-3590; E-mail: jolinda.traugh{at}ucr.edu.

    ABBREVIATIONS

The abbreviations used are: PAK, p21-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
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ABSTRACT
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