The Mechanism of PAK Activation

AUTOPHOSPHORYLATION EVENTS IN BOTH REGULATORY AND KINASE DOMAINS CONTROL ACTIVITY*

Claire ChongDagger , Lydia TanDagger , Louis LimDagger §, and Edward ManserDagger

From the Dagger  Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609 and the § Miriam Marks Department of Neurochemistry, Institute of Neurology, 1 Wakefield St., University College, London WC1N 1PJ, United Kingdom

Received for publication, October 12, 2000, and in revised form, January 31, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both alpha - and beta PAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK N-terminal regulatory domain play no direct role in activation, a phosphorylation of alpha PAK serine 144 or beta PAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma PAK (2) isoform contains a caspase-sensitive site that results in proteolytic activation of gamma 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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of Mutant PAK cDNAs-- alpha - and beta 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 beta -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 [gamma -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-alpha 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 beta PAK was incubated with 10 µg of GST/Cdc42·GTPgamma S in 50 µl of kinase buffer containing 5 µM [gamma -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).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Conserved Autophosphorylation Sites among Different PAK Isoforms-- Cdc42-mediated alpha PAK activation in vitro is accompanied by autophosphorylation of recombinant alpha PAK on 6 serine residues in the N-terminal regulatory domain and 1 threonine residue within the catalytic domain (11). The gamma 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 (beta PAK/PAK3) particularly with a view to identifying the primary autophosphorylation events. The beta PAK was purified from transiently transfected COS7 cells and activated in the presence of excess Cdc42·GTPgamma S (see "Materials and Methods") but limiting ATP concentration; 2 µM kinase was incubated with 5 µM [gamma -33P]ATP (followed by phosphorylation to completion in 500 µM ATP). Fig. 1A shows the profile of HPLC-separated beta 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 beta PAK occur at residues Ser-50, Ser-139, and Thr-421, corresponding to Ser-57, Ser-144, and Thr-422 in alpha 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 beta PAK (10 µg) was activated in vitro with excess Cdc42·GTPgamma S in the presence of 5 µM [gamma -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 beta PAK autophosphorylation occur flanking the CRIB/KID and in the kinase domain.

In gamma PAK as with alpha PAK, all the non-kinase autophosphorylation sites are serine residues (see Fig. 1B); however, gamma PAK S19 (equivalent to alpha PAK T20) was found to be phosphorylated to the same extent as gamma PAKS20 (24). We have recently shown the latter site to be important for the regulation of Nck SH3 binding (25). Because mutant alpha PAK(T422S) is activated normally by Cdc42-GTPgamma S, but no phospho-threonine is detected (Fig. 2A) is seems alpha 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/alpha 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·GTPgamma S (in 25 µl) and myelin basic protein (5 µg). The reaction was initiated by addition of 10 µM [gamma -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 alpha PAK and alpha 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·GTPgamma S and 5 µg of substrate under standard conditions with [gamma -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 alpha 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 alpha PAK (n = 3). The right-hand panels shows the Coomassie Blue-stained PAK protein. The slower migrating species reflect autophosphorylation of PAK.

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 alpha PAK Thr-422, probably in "trans" (23, 26). However, although the T422A mutant exhibited minimal activity toward substrate, it underwent robust autophosphorylation with Cdc42-GTPgamma 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 alpha 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 alpha PAK(L107F, T422A), which lacks a functional kinase inhibitory domain (KID), was compared with alpha 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 alpha PAK(L107F) protein by only ~50%. Thus phosphorylation events in the N terminus, as indicated by the significant mobility shift of the alpha 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 alpha PAK(L107F).


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Fig. 3.   N-terminal phosphorylation events affect PAK activity. A, wild type, alpha PAKL107F or alpha 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 [gamma -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 alpha 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 alpha 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 beta PAK activity as in C.

A Conserved Serine in the KID Modulates PAK Activity-- To investigate further the role of individual autophosphorylation sites, additional alpha - and beta PAK mutants were generated in which the respective serine and threonine were changed to alanine. The closely spaced sites in alpha 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 alpha PAK (activity designated 100%). The alpha PAKS57A and alpha PAKS198A/S203A mutants were activated to the same extent as wild type alpha PAK, whereas the purified S144A/S149A kinase was less active. Similarly purified beta PAK mutants beta PAKS50A and beta PAKS200A were as active as wild type, indicating these residues play no direct role in kinase activation. The beta PAKS139A and beta PAKT421A proteins (equivalent to alpha 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 alpha PAKS144A/S149A mutant was somewhat less active than the single beta PAKS139A mutant (but beta PAK does not contain a site corresponding to alpha PAK Ser-149). These results are in agreement with those obtained by in vitro activation of alpha 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/alpha PAK-(83-149) or the larger Cdc42-binding-defective GST/alpha PAK(S76P)-(1-250) proteins completely block activation of alpha - and beta PAKs by Cdc42·GTPgamma S (7). We therefore wished to test directly whether phosphorylation of the inhibitor sequence could block its function. Although GST/alpha 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/alpha 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 alpha 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 alpha 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 alpha PAK Ser-144 or the corresponding beta 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/alpha PAK-(1-250) containing the S76P substitution (7) was phosphorylated by incubating with purified active alpha PAK and 0.5 mM ATP. After an extensive (60 min) reaction, the kinase was washed out, and GST/alpha 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) alpha PAK-(1-250)-S76P. The activation of alpha PAK by GTPgamma S·Cdc42, which is blocked by alpha PAK-(1-250)-S76P (compare lanes 2 and 3), is essentially unaffected by the phosphorylated inhibitor (lane 4).

Sphingosine Activation of alpha 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 alpha PAK activity in vitro was half-maximal at ~500 µM sphingosine (Fig. 5A). alpha PAK activation was poorer at higher concentrations of sphingosine (>1 mM). This inhibitory effect of sphingosine at higher concentrations was demonstrated when pre-activated alpha 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, alpha PAK (100 ng) was incubated in the presence of sphingosine in 20% Me2SO and 5 µg of MBP at 32 °C with 10 µM [gamma -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 alpha PAK pre-activated with 0.5 mM sphingosine (+10 µM "cold" ATP) is inhibited by 5 mM sphingosine when assayed toward MBP (10 µM [gamma -33P]ATP). C, both Cdc42 and sphingosine-mediated PAK activation are blocked by addition of the KID. alpha PAK (100 ng) was activated by Cdc42·GTPgamma S (250 ng) or sphingosine (100 µM) in the presence and absence of 1 µg of GSTalpha PAK-(83-149) kinase inhibitory domain (KID).

Sphingosine- and Cdc42-mediated PAK Activation through Distinct Sites-- The alpha PAK inhibitor polypeptide (residues 83-148) was similarly effective in blocking both sphingosine- and Cdc42-mediated activation of alpha 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 alpha PAK for in vitro activation induced by either sphingosine or Cdc42·GTPgamma S. As in previous observations (Fig. 3) alpha PAKS144A/S149A and alpha PAKT422A proteins were activated to a lesser extent in vitro by Cdc42·GTPgamma 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. alpha PAK and various substitution mutants as indicated (100 ng) were assayed with or without Cdc42·GTPgamma 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 alpha PAK in the presence of sphingosine.

Fig. 7A shows how the extent of alpha PAK autophosphorylation appears to differ during activation with either Cdc42·GTPgamma 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 GTPgamma S.Cdc42 was seen in terms of PAK activity (i.e. MBP phosphorylation). Activation using sphingosine micelles (100 µM) with excess Cdc42·GTPgamma 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 alpha 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 alpha PAKI75N and alpha PAKS76P mutants (7), which cannot bind GTPgamma 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 alpha PAK was incubated with Cdc42·GTPgamma 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-alpha PAK antibodies. B, the reaction conditions were as above with the following assay conditions: 100 ng of alpha PAK, 250 ng of Cdc42·GTPgamma 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 alpha PAK mutants are activated by sphingosine. A, HA-tagged wild type alpha PAK (WT) or the substitution mutants I75N or S76P were purified using anti-HA antibodies and incubated with Cdc42·GTPgamma 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha - and beta PAK that two conserved sites are key to regulating activity. The alpha 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 alpha 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 gamma 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·GTPgamma S drives specific phosphorylation of Ser-141, and Ser-165, and a higher levels of Thr-402 phosphorylation. This gamma PAK Ser-141 (analogous to alpha 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 alpha 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.

The autophosphorylation of the regulatory threonine, Thr-402 gamma 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 beta 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 gamma 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 (gamma 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 alpha PAK N terminus, including Ser-144 (or equivalent residues for other isoforms). Because alpha 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 alpha PAK-(83-149) is introduced into cells, endogenous alpha 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 alpha 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.

    ACKNOWLEDGEMENTS

We thank Katrin Rittinger for her help with the molecular modeling of the phosphorylated KID structure. The pGEX-MEKK2-(1-290) plasmid was kindly provided by Jonathan Blank. This work is supported by the Glaxo Singapore Research Fund.

    FOOTNOTES

* This work was supported by the Glaxo Singapore Research Fund.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.

To whom correspondence should be addressed: Tel.: 65-874-3766; Fax: 65-774-0742; E-mail: mcbmansr@imcb.nus.edu.sg.

Published, JBC Papers in Press, February 22, 2001, DOI 10.1074/jbc.M009316200

    ABBREVIATIONS

The abbreviations used are: PAK, p21-activated kinase; KID, kinase inhibitory domain; CRIB, Cdc42/Rac1 interaction/binding; FC, focal complex; PI3K, phosphatidylinositol 3-kinase; MBP, myelin basic protein; GST, glutathione S-transferase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; PIX, PAK interacting exchange factor; DPAK, Drosophila PAK; GIT1, GRK interactor 1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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