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GSK-IMCB Group, Institute of Molecular and Cell Biology, Singapore 117609, Singapore,1 Institute of Neurology, University College London, London WC1N 1PJ, United Kingdom2
Received 5 August 2003/ Returned for modification 23 September 2003/ Accepted 30 January 2004
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ABSTRACT |
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PAK autophosphorylation requires the participation of the GIT N-terminal Arf-GAP domain but not Arf-GAP activity and involves phosphorylation of PAK at residues common to Cdc42-mediated activation. Thus, the activation of PAK at adhesion complexes involves a complex interplay between the kinase, Rho GTPases and protein partners that provide localization cues. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The PIX proteins in turn associate with G protein-coupled receptor kinase-interacting target (GIT1), an Arf GTPase activating protein (GAP) which can bind paxillin through its C terminus. Paxillin binding is required to localize GIT1 and associated proteins to focal adhesion complexes (38). The mammalian GITs are derived from either of two related genes, GIT1 and GIT2 (30) which encode proteins with conserved Arf GTPase-activating protein (ArfGAP), ankyrin repeat, Spa2 homology and paxillin binding domains. Paxillin is an abundant protein that is tyrosine phosphorylated following integrin stimulation by focal adhesion kinase, a kinase that also binds to GIT1 (38). At least one function of GIT1 family proteins (also known as protein kinase linker, PKL, or APP) is to serve as a link between the PAK, PIX, and FCs; GIT probably functions in other contexts, for example as a synaptic component (35). It is suggested that Arf6 is a target of GIT1, with which it colocalizes on the plasma membrane and on recycling endosomes, where it participates in Rac-mediated membrane dynamics (33).
In this paper, we report that GIT1 in cooperation with PIX can robustly activate PAK and that this process can occur in the absence of a direct interaction between GIT1 and PIX. This activity requires the ArfGAP domain of GIT1, but does not require the CRIB domain of PAK that binds to small GTPases such as Rac1 and Cdc42. While overexpression of GIT1 alone can activate PAK to a limited extent, PIX isoforms are not of themselves capable of PAK activation. Because PAK activation leads to dissociation of the kinase from the complex (37), this provides a feedback mechanism to control PAK dynamics. Thus, it appears that these PAK partners are poised to provide kinase activation signals in the absence of small GTPase signaling, and points to the importance of studies addressing the temporal and spatial activation of these kinases.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammalian and bacterial expression constructs.
Plasmid pXJ-GST-PAK, pXJ-Flag GIT1, and pXJ-hemagglutinin ß1PIX are as described previously (25, 38). Plasmids encoding GIT1 and ß1PIX carrying various mutations were constructed using a QuikChange site-directed mutagenesis kit (Stratagene) under the manufacturer's conditions. Each new mutant plasmid was sequenced at the Institute of Molecular and Cell Biology central sequencing facility. GIT deletion mutants were generated using internal restriction sites in the cDNA. The N terminus deletion 
1-118 (1ArfGAP) corresponded to the HindIII-KpnI fragment, and 1-375 (GIT1 C) was cloned as a XhoI-KpnI fragment. The C terminus deletion mutant 647-770 was constructed using a BamHI-SmaI fragment from the original GIT1 expression plasmid. For internal deletions, oligonucleotides were designed to amplify corresponding N- and C-terminal fragments of the protein spliced using an introduced unique restriction site. GIT1 lacking the coiled-coil domain lacked residues 424 to 480. The mammalian expression vector allows for in vitro translation using T7 RNA polymerase in rabbit reticulolysate (Promega).
Kinase assays.
Each kinase reaction contained 50 µl of Sepharose immobilized with glutathione S-transferase (GST)-PAK or Flag-PAK, containing 10 µCi of [
-33P]ATP, 10 µM ATP, and 10 µg of GST-substrate protein in kinase buffer (50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM MnCl2, 1 mM dithiothreitol, 0.05% Triton X-100). The mixture was incubated at 30°C for 30 min. Samples were resolved by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE) and processed for autoradiography. The peptide substrate (fused to GST) consists of two tandem repeats of the sequence GDKRDSMVGAP in which the aspartic acid at position –1 confers specificity towards PAK. The reactions were stopped by adding SDS-PAGE loading buffer and heating for 3 min at 95°C. Proteins were resolved by SDS-9% PAGE and visualized by PhosphorImager analysis (Amersham Biosciences) prior to immunoblotting.
Cell transfection, fractionation, and immunoprecipitation.
Relevant cDNAs were cloned in pXJ-Flag, pXJ-Ha or pXJ-GST mammalian expression vectors (23). COS-7 cells at 70 to 80% confluency (60-mm-diameter culture dish) were starved 1 h with serum-free Dulbecco's modified Eagle medium (SF-DMEM) prior to addition of DNA-Lipofectamine (Gibco BRL). For each dish 4 µg of total plasmid DNA (in 200 µl of SF-DMEM) was mixed with 25 µg of Lipofectamine reagent and incubated for 30 min at room temperature. This mix was diluted with 1.6 ml of SF-DMEM and added to the cells (60-mm-diameter dish). After 3 h fetal bovine serum was added to a 1% final concentration. NIH 3T3 cells were transfected using a standard calcium-phosphate precipitate protocol and harvested 40 h afterwards. Transfected cells were harvested by scraping in ice cold cell lysis buffer (500 µl of 50 mM HEPES [pH 7.3], 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 20 mM ß-glycerophosphate, 5% glycerol, 1% Triton X-100, 1 mM dithiothreitol, and a protease inhibitor cocktail [Roche]). Cells were broken by 10 passages through a 29-gauge insulin syringe before clarification at 14,000 x g for 10 min. Pellets were washed with lysis buffer for 15 min on ice, repelleted, and suspended in appropriate volume of 1x SDS sample loading buffer. For SDS-PAGE samples were heated (3 min at 95°C), run immediately, and transferred to polyvinylidene difluoride membranes for Western blot analysis. For immunoprecipitation 200 µl of lysate was passed through 25 µl of anti-Flag M2 Sepharose, and the beads were washed with 1 ml lysis buffer. For GST-PAK purification, 400 µl of lysate purified using 50 µl of glutathione agarose (Pharmacia) an the amount of protein recovered quantified by Coomassie staining or using anti-PAK antibodies.
Immunocytochemistry, microscopy and image processing. HeLa cells were plated on coverslips in 30-mm-diameter culture dishes and grown to 60% confluency. Plasmid DNA (1 µg per 5 µl of Lipofectamine reagent) was mixed in 200 µl of SF-DMEM for 30 min and then diluted to 1 ml for addition to cells. After 2 h, fetal bovine serum (Gibco BRL) was added to 1% final concentration. After 16 h cells were fixed in 3% paraformaldehyde for 5 min and permeabilized (10 min) with phosphate-buffered saline (PBS) containing 0.5% Triton X-100. Primary antibodies were added (100 µl per coverslip in PBS plus Triton X-100). Primary antibodies were incubated 2 h (30°C) on the coverslips. Secondary fluorescent antibodies (Molecular Probes) were incubated for 1 h before washing in PBS containing 0.1% Triton X-100. Secondary antibodies were coupled with Alexa 488 or Alexa 546 dyes (Molecular Probes). For fluorescence observation these secondary antibodies were visualized on a Zeiss Axioplan II microscope using a Plan-Apochromat 63x/1.40 objective, and images were collected on a Coolsnap HQ charge-coupled device.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously showed PIX to be important for the focal adhesion localization of PAK (25). Others subsequently showed that for correct focal adhesion localization of PIX, its interaction with GIT1 interaction is required (6). Unlike wild-type GIT1(Fig. 2A, upper panels), GIT1 M4 was poorly localized to focal adhesions (lower panels), although it had an intact paxillin binding domain (residues 646 to 770). Since the latter domain suffices to target GIT1 to focal adhesions (21), this poor localization of M4 supports our proposal that the PIX/GIT1 interaction drives the paxillin association (38). High-resolution images of GIT1 or PIX costained with paxillin (Fig. 2B) show the distribution of GIT1 and PIX to be asymmetric within focal adhesions, with PIX and GIT1 being enriched proximal to the cell edge side of adhesion complexes (Fig. 2B). By contrast GFP-paxillin and vinculin are perfectly colocalized in the same cell (data not shown). This asymmetry of distribution maybe a useful means of identifying other proteins associate with the GIT1/PIX complex.
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and ß PIX isoforms (16). The corresponding Drosophila dPIX region is more divergent (Fig. 3A), although likely to bind dGIT1 in an analogous manner. This N-terminal limit of the GIT1 binding region (13) is boxed; because this region is predicted to adopt an alpha-helical fold, residues I539/E540 were replaced with the "helix breakers" glycine and proline, respectively. This mutant, like Cool-1 V538A (13), failed to coprecipitate with GIT1 (Fig. 2B). The disposition of GFP-PIX(I539P/E540G) in HeLa cells was as expected: while wild-type PIX associated with punctate focal adhesions, the mutant did not (data not shown).
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PAK species. Because of this effect under these conditions we used the PIX(I539P/E540G) mutant for many experiments in which we wished to recover the majority of GST-PAK for further analysis.
GIT1 but not PIX activates PAK.
PAK is largely inactive when expressed in mammalian cells although the kinase is activated in bacteria through an undefined mechanism (23). Coexpression of GIT1 with PAK (Fig. 4A) clearly increased PAK activity (lanes 2). GIT1(1-646) lacking the paxillin -binding domain and the D294K/E295R (M4) mutant were equally effective in increasing PAK activity, suggesting neither paxillin nor ß1PIX-binding was necessary for GIT1-induced activation of PAK. Since GIT1 could activate PAK, we were interested in whether the PAK partner ß1PIX was involved. PIX is characterized by two isoforms (PIX and ßPIX) with the latter exhibiting many alternate spliced versions (33); ß1PIX is the predominant form in cultured cells such as HeLa and fibroblast lines (16). It has been reported that PIX but not ß1PIX (Cool-1) activates PAK (13). We therefore tested the studied effects of PIX alone (Fig. 4B): PAK autophosphorylation was assessed by phospho-specific antibodies targeted to known sites (23). In common with many kinases phosphorylation within the activation loop (T422 in PAK) is critical for catalytic activity towards substrates (7, 34). Unlike GIT1 no full-length isoform of PIX that we tested was capable of driving PAK autophosphorylation.
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PAK is the predominant isoform). Cell lysates were used for anti-PAK immunoprecipitation and assessment of PAK activity as well as levels of associated PIX and GIT1 (Fig. 5A). Both of these proteins were enriched to a similar level to PAK in the immunoprecipitate relative to input (lane 1) indicating a significant proportion of cellular PAK is part of the trimeric complex. Both wild-type GIT1 and GIT1(1-504), which cannot associate with adhesion complexes, caused an increase in PAK activity relative to the mock-transfected control (lane 2 and 3). Increasing the level of GIT1 increased the yield of PAK-associated GIT1 (lane 4), but the increased activity does not appear to result from adhesion complex targeting per se since GIT1(1-504) had a similar effect.
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PIX or ßPIX (Fig. 5B lanes 5 and 6). Expression plasmids were transfected and total cell lysates then probed with antibodies against GIT1, PIX and PAK, as well as phospho-specific antibodies to the pS198/203 and T422 sites on PAK. The exogenous levels were estimated as 
18-fold higher for GIT1 and 12-fold higher for ßPIX based on densitometry of the Western blot bands (top panels). Hyper-phosphorylated forms of PAK are evident from the anti-PAK Western blot: inclusion of the two PIX isoforms caused a more complete shift in PAK and some increase in pS198 relative to that with GIT1 alone.
The ArfGAP domain of GIT1 is required for PAK activation.
To further analyze PAK activation by GIT1 while avoiding translocation of PAK to the Triton X-100 insoluble fraction (Fig. 3), the ß1PIX I539P/E540G was used since this mutant complexes to PAK (via its SH3 domain) and remains as a homodimer (via the coiled-coil C-terminal domain) but behaves similarly to wild-type PIX terms of kinase activation when expressed with GIT1. Analysis was performed using transient transfection of plasmids encoding GST-PAK, Flag-GIT1, and Ha-ß1PIX. GIT1 expression induced PAK autophosphorylation at residues S144 and S198/203 more robustly than for T422. However GIT1 constructs alone did result in significant phosphorylation at T422 with longer exposure times (Fig. 5B). Various GIT1 constructs (C1 to C5) were used to assess the GIT region required to cause PAK autophosphorylation. The GIT1 leucine zipper deletion mutant (C2) is expected to remain monomeric (15, 28) but was reasonably active in terms of its effect on PAK. Notably, constructs encoding GIT1 N terminus deletion mutants (C3 and C4) that likely affect its interaction with Arfs failed to activate PAK. The deletion of amino acids 1 to 118 removes the ArfGAP domain (schematically shown in Fig. 6). The amount of ß1PIX copurified with PAK (as shown) did not significantly vary with the activation status of PAK (cf. lane 3 versus lane 9). In subsequent pull-down experiments, we often used GIT1(1-504) as an activator since it was well separated from the PIX (78 kDa) and GST-PAK (95 kDa).
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PAK.
Since a critical region for PAK activation lay within residues 1 to 117, two mutants were generated in a GIT1(1-504) background to address the role of the ArfGAP domain (Fig. 7A). The C11/14G mutant (changing the Zn2+ coordinating cysteines) which disrupts the zinc finger is expected to cause localized protein misfolding, while the R39K mutant is anticipated to abolish ArfGAP activity only (22). The R39K mutant appeared to be as effective as the wild type in promoting PAK autophosphorylation (Fig. 7B), while the GIT1(C11/14G) mutant was not. Thus, the ability of GIT1 to drive PAK autophosphorylation in the presence of PIX depends on the structural integrity of the Arf GAP domain but does not require GAP activity per se.
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PAK autophosphorylation.
PAK activation need not require Cdc42 and Rac1 binding.
Because ß1PIX is an activator of Rac1 (25), we next examined whether GIT1/PIX acted on PAK via Cdc42 or Rac1 binding through the regulatory domain. The experiments involved cotransfection of dominant-inhibitory mutants (T17N) of Cdc42 and Rac1, or use of a PAK mutant that cannot interact with these GTPases (Fig. 8). We show that neither T17N GTPase mutant blocked PAK activation by GIT1/PIX as assessed by T422 phosphorylation (Fig. 8A, left panel). Most surprisingly, PAK(S76P), which was nonresponsive to Cdc42G12V (Fig. 8A, right panel, lane 4), underwent robust autophosphorylation when cotransfected with GIT and PIX (last lane), indicating the activation mechanism is unlikely to involve any GTPase of the Rho family.
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PAK N terminus contains the binding site for the second SH3 domain of Nck (2, 5, 14, 37). As shown in Fig. 8B, a deletion construct (PAK N22) lacking the Nck-binding site was similarly activated (as assessed by mobility shift) as for Cdc42-mediated activation. The mechanism of GIT1 activation is conserved among PAK isoforms, since ßPAK behaved in an identical manner to PAK. It seems likely that the three conventional PAK1/2/3 isoforms can be activated via associated PIX and GIT.
Activation of PAK at adhesion complexes.
Because transient expression experiments were performed 16 to 20 h after DNA addition there remained is issue of whether GIT1 could lead to acute activation of PAK. To assess this we microinjected NIH 3T3 cells with plasmids encoding Cdc42V12 or GIT1 (Fig. 9A, upper and lower panels). After 2 h autophosphorylated kinase was detected at adhesion complexes (Fig. 9A), which are predominantly peripheral with Cdc42. The neither anti-pT422 nor anti-pS144 antibody detected the active PAK after cell fixation.
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PAK at these sites. We then looked at the disposition of PAK in the presence of GIT1(C11/14G), which is defective for PAK activation (Fig. 6). Cells expressing low levels of GFP-PAK and this GIT1 mutant indeed showed clear focal adhesion localization (Fig. 9B). This was comparable with the localization of PAK when coexpressed with the kinase-inhibitory domain (KID) (right panel). These experiments explain why we fail to detect stable association of PAK with PIX in RhoA-dependent adhesion complexes: although these structures may not contain active Cdc42 or Rac1 the GIT/PIX complex of itself drives activation which leads to the consequential loss of the PAK.
Concentration-dependent autoactivation of PAK.
Since the CRIB region is not implicated in PAK activation, we sought other conserved regions in surrounding sequences that might play a role in disinhibiting PAK in the presence of GIT1. A crystal structure of the autoinhibited catalytic domain of PAK unfortunately does not include residues 1 to 69 and 150 to 248 (19), which may play additional roles in kinase activation. However, various strategies to identify the residues involved in the GIT1-mediated PAK activation failed. During these experiments, we uncovered a property of PAK that could provide the means for its activation in vivo. When PAK is immobilized at higher concentration and incubated with Mg2+-ATP it is able to undergo a GTPase-independent activation. As shown in Fig. 10A, over a relatively modest concentration range (from 0.5 to 2.5 µM) the protein behaves dramatically differently with respect to autoactivation. These samples were normalized with respect to protein loading per lane after elution from the matrix (lower panel). In Fig. 10B the CRIB mutant S76P PAK demonstrated the same behavior but is refractory to Cdc42 activation at the lower concentration. Thus, a local recruitment of PAK may predispose the kinase to activation in vivo. This effect can explain why membrane-targeted PAK is active (20) as well as the generation of predominantly active kinase in Escherichia coli (23).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The targeting of GIT1-ß1PIX to focal complexes is likely to be intimately linked to PAK kinase activation since ß1PIX recruits PAK to focal complexes (25). In a different context, Drosophila dPak was found to be mislocalized at synapses in dPIX mutant flies (29). By contrast, dPIX was localized normally in Pak mutant strains underlining that the kinase requires its partner dPIX to localize correctly. Interestingly Rac1-PAK signaling is regulated in a spatial manner; Rac1-GTP only couples to PAK on the plasma membrane in adherent cells (9). In this case, it appears the sequestration of active Rac1 by RhoGDI at internal sites prevents interaction with effectors such as PAK but the mechanism of integrin-mediated translocation of PAK has not been established.
Domains involved in GIT1/ß1PIX targeting to focal complexes.
We and others have observed that GIT1 and ß1PIX proteins are enriched in focal complexes induced by Rac1 or Cdc42 (6). In RhoA-type focal adhesions, the PAK-associated complex of GIT and PIX appears to be destabilized by the activity of PAK (6) which is blocked by the KID of PAK. In transfection experiments, only inactive kinase was found to be enriched in these structures (23). In most cultured cells, integrin-dependent focal adhesions are predominantly of the RhoA-type and unaffected by introduction of dominant inhibitory Rac1 or Cdc42. The point mutants of GIT1 and ß1PIX that prevent their mutual association enable us to probe the role of this interaction. Neither GIT1(D294K/E295R) nor ß1PIX(I539P/E540G) localizes significantly to FCs. However, even if incapable of associating with FCs, both mutants would be expected to oligomerize with the endogenous wild-type protein, allowing (weaker) indirect FC localization. That GIT1(D294K/E295R) is so poorly FC-localized indicates ß1PIX provides an input to allow association with focal adhesions: one possibility is the up-regulation of GIT1-paxillin interaction by ß1PIX (38).
Aside from the presence of GIT1 at adhesion complexes the protein may reside in a specific endo-membrane compartment (26) or form insoluble cytoplasmic protein complexes (21). It is not clear at this stage what protein or lipid interaction underlies this localization, and is the subject of ongoing studies.
PIX and GIT1 as mediators of PAK activation.
Previous studies of PAK activation in vivo have focused on PIX, as it was shown that ß1PIX promoted GTP loading and activation of Rac1 (25). Since Cdc42(G12V/Y40C), which lacks an ability to bind and activate PAK, was synergistic with ß1PIX in PAK activation, a model was proposed whereby ß1PIX recruits PAK for activation by Rho GTPases such as Rac1 (generated by guanine nucleotide exchange factor activity of ß1PIX). A truncated form of ßPIX (residues 155 to 545) apparently activated PAK1 whereas full-length ßPIX had no effect (8), which is line with our data (Fig. 6). These observations are complicated by the fact that PIX can also recruit negative regulators such as the type 2C phosphatase POPX (17). We have looked for the interaction between GIT1 and a variety of other protein phosphatases (data not shown), since GIT1 might activate PAK by sequestering a different phosphatase. Although we demonstrate no requirement for direct interaction between GIT1 and PIX to drive this PAK activation, it is clear in vivo that it is their mutual interaction that leads to accumulation of both proteins at adhesion complexes (cf. Fig. 2). Our data provide two important insights: firstly that microenvironments in the cell where PIX and GIT1 accumulate are sites of PAK activation regardless of Rho GTPase status: secondly that elevated levels of GIT1 or increased association of PAK with the GIT/PIX complex in cells is likely to be associated with increased kinase activity.
There has been no previous report that GIT1 increases PAK activity; GIT1 (CAT1) and GIT2 (CAT2) being reported not to affect Pak3 (ßPAK) activity (1). Full-length GIT1 has similar effects on PAK as the C-terminal truncated GIT1(1-504) (Fig. 6). Surprisingly, the ArfGAP domain of GIT1 plays a key role in activation of PAK, with structural integrity of this domain rather than GAP activity being important. GIT1 has potential as an Arf6 effector signaling to PAK; however we have been unable to enhance the GIT activation of PAK with active Arf6 (data not shown).
Three lines of evidence indicate that GIT1's effect on PAK is independent of the binding of GTPases such as Cdc42 or Rac. Firstly, PAK activation was not driven by ßPIX alone (i.e., Rac1 activator); secondly, dominant inhibitory Cdc42 or Rac1 mutants failed to block the GIT1 effect; thirdly and most telling, the PAK(S76P) mutant which cannot bind the small GTPases was activated by the PIX-GIT1 combination. This activation mechanism was tested for two rat isoforms, - and ßPAK (i.e., equivalent to human PAK1 and PAK3), and did not require the N terminus that binds to Nck.
The translocation of cytoplasmic proteins to new sites of action is a common theme in signal transduction. Such mechanisms are likely to increase the local concentration of proteins and facilitate interaction with targets. What is interesting about the autoactivation of PAK is that the kinase has already been shown to exist as an autoinhibited dimer (19); if dimer "breathing" were a mechanism that allowed autoactivation, then increasing PAK concentration should counter this tendency. PAK has been shown to be activated by a variety of lipids in vitro, including sphingosine and phosphatidic acid (5, 7); thus, these are candidate intermediaries for the GIT1/PIX effect, but to date we have been unable to derive PAK mutants that are lipid insensitive.
In conclusion, our data suggest a model in which the GIT1/PIX complex serves as a docking site for PAK at adhesion complexes which leads to the activation of PAKs regardless of the local activation status of Cdc42 or Rac1. They also indicate that PAK activity is dependent on physiological levels of GIT1 and PIX.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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