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Kinases are important therapeutic targets in oncology due to their frequent deregulation in cancer. Typical ATP-competitive kinase inhibitors, however, also inhibit off-target kinases that could lead to drug toxicity. Allosteric inhibitors represent an alternative approach to achieve greater kinase selectivity although examples of such compounds are few. Here we elucidate the mechanism of action of IPA-3, an allosteric inhibitor of Pak kinase activation. We demonstrate that IPA-3 binds covalently to the Pak1 regulatory domain and prevents binding to the upstream activator Cdc42. Pre-activated Pak1, however, is neither inhibited nor bound significantly by IPA-3, demonstrating exquisite conformational specificity of the interaction. Using radiolabeled IPA-3 we show that inhibitor binding is specific and reversible in reducing environments. Finally, cell experiments using IPA-3 implicate Pak1 in phorbol-ester stimulated membrane ruffling. This study reveals a novel allosteric mechanism for kinase inhibition through covalent targeting of a regulatory domain.
Protein kinases have attracted much attention as therapeutic targets in oncology due to their frequent dysregulation in cancer and their tractability to small-molecule inhibition. A significant challenge in the development of kinase inhibitors is achieving kinase selectivity, due to the evolutionary conservation of the ATP-binding pocket among kinases. An attractive alternative approach would be allosteric inhibition by compounds binding at distant, less conserved sites. Kinases frequently contain regulatory domains that regulate kinase location and/or catalytic activity and these domains may offer alternative targets for allosteric kinase inhibition.
IPA-3 is a non ATP-competitive, allosteric inhibitor of p21-activated kinase 1 (Pak1) (1), a cytoplasmic serine/threonine kinase whose hyperactivity has been closely linked to tumorigenesis (reviewed in (2–5)). Pak1 contains an N-terminal regulatory domain (RD) that in the inactive, homodimeric state binds and inhibits the catalytic activity of the C-terminal kinase domain of its partner and vice versa (Figure 1A). Binding of the p21 GTP binding proteins Rac or Cdc42 to the RD displaces the RD from the catalytic domain, dissociating the dimer and leading to kinase activation (6, 7). IPA-3 was identified in an in vitro screen for compounds that inhibit Pak1 activation by Cdc42 (1) but the mechanism by which IPA-3 inhibits Pak1 activation is unknown. Nevertheless, a variety of studies have begun to utilize this compound to investigate Pak functions in cells (1, 8–10). Here, using a multidisciplinary approach including fluorescence spectroscopy, affinity precipitation, kinase assays and binding assays using radiolabeled IPA-3, we show that IPA-3 inhibits Pak1 activation in part by binding covalently to the regulatory domain of Pak1. This work illustrates a novel approach to kinase inhibition that provides significant target selectivity and implicates Pak1 as a critical downstream effector of protein kinase C in actin cytoskeletal rearrangements.
Cdc42-GTPγS, full-length Pak1, activated Pak2, GST-Rac1, GST-HR1 (from PRK1), MBP and GST-mini-N-WASP were prepared as described (11–14). GST-Pak1 kinase domain K299R (amino acids 248–545) and GST-RD (amino acids 67–150) were generated by PCR from templates provided by J. Chernoff and were cloned into pGEX-6P1 and purified from E. coli. Plasmids encoding HA-tagged human Pak1 (in pJ3) and myc-tagged Cdc42 (in pCMV6M) were generously provided by J. Chernoff.
Emission spectra (excitation 280 nm) were recorded with a Cary Eclipse (Varian) in Kinase buffer (50 mM HEPES, pH 7.5, 12.5 mM NaCl, 0.625 mM MgCl2, 0.625 mM MnCl2) at 22°C. A 0.5 μM solution of each protein was titrated in parallel with IPA-3 or dimethyl sulfoxide (DMSO, solvent control) and, after 5 minute incubation, tryptophan emission was monitored at 340 nm. Fluorescence changes observed in DMSO only titrations were subtracted to remove solvent effects on tryptophan fluorescence.
Pak1 (150 nM final) was pre-incubated with MBP (8.3 μM), indicated proteins, and IPA-3 or DMSO in Kinase buffer for 20 minutes at 4°C. Cdc42-GTPγS (3.2 μM) was then added and the reaction was pre-equilibrated 10 minutes at 30 °C. Kinase reactions were started by the addition of ATP (to 30 μM) containing [32P]ATP and were incubated 10 min and analyzed by SDS-PAGE and autoradiography.
10 μg GST or GST-RD or 20 μg GST-mini N-Wasp bound to Glutathione Sepharose 4B were incubated with IPA-3 or PIR3.5 for 10 minutes at room temperature and then 30 minutes on ice in 100 μL Binding buffer (50 mM Hepes pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1% Nonidet P40). Cdc42-GTPγS (5 μg) was added for 30 minutes. Beads were washed with Binding buffer and resuspended in sample buffer for SDS-PAGE and western blotting with anti-Cdc42 antibodies (Santa Cruz Biotechnology).
For cell studies, HEK293 cells were transfected with myc-Cdc42 or HA-Pak1 using Lipofectamine 2000 (Invitrogen) and the following day serum was withdrawn from the culture medium. 48 hours after transfection, cells were treated with DMSO (solvent control) or 50 μM IPA-3 for 10 minutes prior to addition of 250 ng/ml PMA for 15 minutes. Cells were lysed in cold lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 10 % glycerol, 1% Nonidet P-40, 50 mM NaF, 5 mM Na3VO4) and clarified lysates were immunoprecipitated with anti-myc (Santa Cruz Biotechnology) or anti-HA (Covance) antibodies and analyzed by SDS-PAGE/western blotting for myc or HA.
14C radiolabeled IPA-3 (2837 ± 85 cpm/nmol) was synthesized essentially as previously described (1) except using 8-[14C]-labeled 2-naphthol (Sigma) as a precursor. Details and product characterization are available as Supporting Information. For autoradiographic detection of [14C]-IPA-3, dried SDS-PAGE gels were exposed to BioMax MS film (Kodak) using a BioMax TranScreen-LE (Kodak). Exposure times were typically 7 days.
Pak1 or other proteins were incubated with [14C]-IPA-3 for 1 hour (or as indicated) in Kinase buffer at 30°C. Covalently bound [14C]-IPA-3 was separated from free [14C]-IPA-3 by precipitation using 10 volumes of acetone and incubation for 1 hour at 30°C. Precipitates were recovered by centrifugation, solubilized in 2% SDS and analyzed by scintillation counting (Beckman LS6000SC). Efficiency of protein precipitation was > 90% as assessed by silver staining of SDS-PAGE gels of precipitated protein from a parallel experiment using unlabeled IPA-3.
Covalent binding of [14C]-IPA-3 was also assessed by their co-migration in non-reducing SDS-PAGE. For Figure 4B, ,1.11.1 μg recombinant Pak1 was first added to serial dilutions of a high speed supernatant of Xenopus egg cytoplasmic extract (7.2 mg/mL) prepared as described but without DTT (12) and then [14C]-IPA-3 was added to 20 μM. After a 1 hour incubation at 30 C, samples were analyzed as above.
For Figure 3D, 15 μM [14C]-IPA-3 was incubated with pure Pak1 for 1 hour at 30°C in Kinase buffer and then partially proteolyzed for the indicated times on ice with 2.2 μg/ml chymotrypsin to generate chymotrypin-resistant core fragments (15) and analyzed by 15% SDS-PAGE and autoradiography.
BS-C-1 cells were pre-treated for 10 minutes with either DMSO, control compound PIR3.5 or IPA-3 (50 μM). Cells were then stimulated for 15 minutes with 250 ng/ml phorbol myristate actetate (PMA) in culture medium and fixed in phosphate-buffered saline containing 4% formaldehyde for 20 minutes at room temperature. For the washout experiment, cells were treated for 10 minutes with 50 μM IPA-3 and then washed into media without IPA-3 but containing 500 μM cycloheximide for 1 hour prior to stimulation with PMA and fixation as above. After permeabilization, filamentous actin was stained with Alexa 488-phalloidin and DAPI (Molecular Probes) and quantified as described (16). For Pak1 localization, BS-C-1 cells were transfected with HA-Pak1 using Lipofectamine 2000 (Invitrogen). Transfected Pak1 was localized in PMA-stimulated cells by indirect immunofluorescence using an anti-HA antibody (Covance).
IPA-3 (Figure 1B) inhibits the activation of Pak1 by small GTPases but does not inhibit the catalytic activity of pre-activated Pak1 (1). These observations suggest that IPA-3 might perturb conformational changes that normally accompany Pak1 activation. To determine whether IPA-3 interacts with the Pak1 RD, we titrated a solution of full-length Pak1, truncated Pak1 fragments, or control proteins with IPA-3 and monitored changes in intrinsic tryptophan fluorescence. Three tryptophan residues are located within the kinase domain and one within the RD. Saturable binding of IPA-3 to full-length Pak1 was observed with an apparent dissociation constant of 1.92 ± 0.2 μM, consistent with the reported IC50 of 2.5 μM (Figure 1C). IPA-3 bound the isolated RD with an even higher apparent affinity (0.1 ± 0.01 μM) whereas the kinase domain showed a weaker interaction. As negative controls, GST alone and free tryptophan showed only weak perturbation of fluorescence. These observations indicate a direct binding of IPA-3 to the RD of Pak1. In addition, the higher apparent affinity for the isolated RD compared to full-length Pak1 suggests that IPA-3 may bind to a conformation of the RD distinct from that found in autoinhibited Pak1.
Next we reasoned that if IPA-3 binds the Pak1 RD, then excess RD might titrate IPA-3 away from full-length Pak1 in vitro, protecting full-length Pak1 from inhibition by IPA-3. To test this, Pak1 and MBP were incubated with or without GST-RD or GST and then IPA-3 was added (or not). Cdc42-GTPγS (hereafter simply “Cdc42”) and [32P]-ATP were added to initiate the kinase assay. As expected, Cdc42 promoted Pak activation leading to higher levels of MBP phosphorylation and this increased kinase activity was inhibited by IPA-3 (Figure 1D; compare autoradiogram lanes 1–3). Inclusion of GST-RD protected Pak1 from IPA-3-mediated inhibition in a dose dependent manner (lanes 4–5) whereas GST alone did not (lanes 8–9). This finding is particularly striking since GST-RD on its own inhibits Pak1 activation (compare lanes 6 & 7 with lane 2) by both sequestering Cdc42 and by directly inhibiting Pak1 (15). Together, our results demonstrate direct binding of IPA-3 to the RD of Pak1.
Binding of small GTPases to the Pak1 RD initiates conformational changes leading to Pak1 activation. Since IPA-3 also binds to the RD, we tested whether IPA-3 inhibits Cdc42-RD binding. GST-RD immobilized on beads was used to precipitate soluble Cdc42 in the presence or absence of IPA-3. IPA-3 inhibited the interaction of Cdc42 with the Pak1 RD in a dose-dependent manner (Figure 2A; lanes 3–5). As expected, the structurally related but inactive compound PIR3.5 had no effect (lanes 6–8). Moreover, IPA-3 has no effect on the binding of Cdc42 to the homologous RD from N-WASP (lanes 9–10), which shares 30% sequence identity to the RD of Pak1. Thus IPA-3 selectively prevents Pak1 activation, in part, by preventing its interaction with small GTPase activators.
To confirm that IPA-3 could disrupt binding of Cdc42 to full-length Pak1 in the cellular context, we transfected HEK293 cells with Cdc42 and Pak and monitored their association by co-immunoprecipitation in the presence or absence of IPA-3 (Figure 2B). Treatment with IPA-3 inhibited the binding of Pak1 to Cdc42 (compare lanes 2 and 3), supporting the in vitro results.
IPA-3 contains a disulfide bond, suggesting that it might act through covalent redox modification of Pak1. We previously showed that IPA-3 does not form mixed disulfides with surface-exposed cysteine residues of Pak1 (1). To test whether IPA-3 might covalently modify Pak1 at other sites, we synthesized IPA-3 using 8-[14C]-labeled 2-naphthol as a precursor to introduce the radiolabel into both naphthol ring systems (see Figure 1B). Increasing amounts of recombinant Pak1 were incubated with [14C]-IPA-3 under conditions in which IPA-3 inhibits Pak1 activation by > 90% and then Pak1 protein was acetone precipitated under denaturing conditions. Whereas [14C]-IPA-3 alone was freely soluble in acetone (not shown), a small fraction of [14C]-IPA-3 precipitated in a Pak-dependent manner with a stoichiometry of ~2.5 mol IPA-3/mol Pak1 (Figure 3A, squares). Quantitatively similar binding was observed to the truncated constructs comprising the RD and kinase domains. No binding was detected to GST alone, however. Notably, the Pak1 RD lacks any cysteine residues, indicating that IPA-3 binding to this domain does not occur through the formation of a mixed disulfide. Time-dependent inhibition is a hallmark of covalent inhibitors and kinetic experiments using this assay revealed slow binding of IPA-3 to Pak1 with a saturating stoichiometry of 2.6 +/− 0.5 mol IPA-3/mol Pak1 (Figure 3B).
To confirm that IPA-3 binding is covalent, Pak1 or GST were incubated with [14C]-IPA-3 and then analyzed by non-reducing SDS-PAGE and autoradiography. Co-migration of the radiolabel with Pak1 confirmed covalent binding of IPA-3 to Pak1 whereas no binding of [14C]-IPA-3 to GST was detected (Figure 3C, upper panels). [14C]-IPA-3 also bound fragments of Pak1 corresponding to the RD and a catalytically dead form of the kinase domain, though with a higher apparent affinity to the RD (lower panels).
To determine where IPA-3 bound in the context of the native homodimer, full-length Pak1 was incubated with [14C]-IPA-3 followed by limited proteolysis with chymotrypsin, which produces protease-resistant fragments corresponding to the core of the regulatory and kinase domains (15). SDS-PAGE and autoradiography of the proteolytic digests demonstrated radioactivity associated with both core domains (Figure 3D). Phosphor imager analysis of the proteolytic digests demonstrated ~ 37% of the Pak1-bound IPA-3 was associated with the stable catalytic domain (Figure 3D, lower panel), implying that the remaining radioactivity is present in low molecular weight peptide fragments which would include the RD. Thus, although IPA-3-RD binding prevents Cdc42-RD binding, we cannot rule out additional affects mediated by IPA-3 bound to the kinase domain. Though if important for Pak inhibition, this binding site must only appear only transiently during activation since catalytically active Pak1 is not inhibited by IPA-3 (1).
We predicted that, if covalent, binding of IPA-3 to Pak1 should be temperature-dependent and irreversible under our in vitro conditions. To test this prediction, we pre-incubated [14C]-IPA-3 with Pak1 and MBP at 4°, 15° or 30°C and then added excess RD to sequester residual free IPA-3. The temperature of all reactions was then shifted to 30°C, Cdc42-GTPγS and [32P]ATP were added and Pak1 kinase activity was measured. Pak1 pre-incubated with IPA-3 at 4°C prior to RD addition showed robust kinase activity due to dose-dependent sequestration of IPA-3 by RD (Figure 3E, compare lane 3 to lanes 4–5). Pak1 kinase activity was progressively inhibited after pre-incubation at 15° or 30°C prior to RD addition (lanes 6–9). This result shows that inhibition by IPA-3 occurs in a temperature-dependent and irreversible manner during the pre-incubation step.
The low stoichiometry and saturability of IPA-3 binding to Pak1 and the lack of binding to GST suggested that the binding, though covalent, was highly specific. Indeed, IPA-3 exhibits significant kinase selectivity and also does not inhibit the catalytic activity of Pak1 that has been pre-activated by Cdc42 (1). We therefore assessed the ability of IPA-3 to bind to Pak1 after pre-activation by Cdc42. Whereas inactive Pak1 bound IPA-3 robustly, binding of IPA-3 to pre-activated Pak1 was substantially reduced (Figure 4A, compare lanes 1 and 2), consistent with the inability of IPA-3 to inhibit pre-activated Pak1.
We also tested the ability of IPA-3 to bind covalently to a variety of other proteins. IPA-3 showed selectivity for inactive Pak1 or RD and did not bind significantly to other tested proteins including bovine serum albumin (Figure 4A). To determine the selectivity of IPA-3 for Pak1 in a more complex protein mixture, recombinant Pak1 was added to serial dilutions of a cytosolic extract of Xenopus laevis eggs and [14C]-IPA-3 was added and the reaction was incubated for 1 hour. Mixtures were then analyzed by non-reducing SDS-PAGE and Phosphorimager analysis. In the most concentrated sample, Pak1 represented only 0.76 % of total protein, yet IPA-3 binding to Pak1 was unaffected by the presence of excess Xenopus proteins and individual radiolabeled proteins other than Pak1 were not observed (Figure 4B). Because of limitations in the sensitivity of detection of [14C]-IPA-3, we cannot conclude that proteins other than Pak1 do not bind IPA-3. Nor would we expect to detect binding of IPA-3 to the low concentration of endogenous Pak1 protein. Nevertheless, the robust and specific labeling of added recombinant Pak1 in this complex mixture supports a selective binding of IPA-3 to Pak1. We thus conclude that the selective kinase inhibitory profile of IPA-3 can be explained by a specific and conformation-dependent binding of IPA-3 to Pak1.
The disulfide bond of IPA-3 is critical for inhibition of Pak1 and in vitro reduction by the reducing agent dithiothreitol (DTT) abolishes Pak1 inhibition by IPA-3 (1). We therefore tested whether IPA-3 bound covalently to Pak1 would be released by DTT treatment. Pak1 was incubated with [14C]-IPA-3 followed by a range of DTT concentrations. Quantitation of Pak1-bound [14C]-IPA-3 from acetone precipitates revealed that DTT treatment released IPA-3 in a dose-dependent manner (Figure 4C).
The cell cytoplasm is a reducing environment, which might result in reduction and inactivation of IPA-3 in cells. Nevertheless, IPA-3 treatment of cells inhibits Pak1 activation (1), most likely because of the large reservoir of freely exchanging IPA-3 present in the oxidizing environment of the cell culture medium. We therefore predicted that IPA-3 inhibition in cells might be reversed by the cellular redox environment on removal of IPA-3 from the culture medium. Pak1 activity is closely linked to dorsal membrane ruffling (17, 18) and stimulation of cells by the protein kinase C agonist phorbol myristate acetate (PMA), activates Pak isoforms (19) and produces actin-rich ruffles containing Pak1 (Figure 5A). PMA-induced ruffling is blocked by inhibitors of Rho GTPase signaling (16) consistent with a role for Pak1. IPA-3, but not the control compound PIR3.5, inhibited PMA-stimulated ruffling (Figures 5B & C). Removal of IPA-3 from the culture media and addition of cyclohexamide to block new protein synthesis restored the ability of PMA-stimulated cells to ruffle (Figures 5B & C). We therefore conclude that the effect of IPA-3 is reversible in live cells, most likely through reductive release of IPA-3.
The present study has elucidated a novel mechanism for kinase inhibition by IPA-3, a non-ATP competitive inhibitor of Pak1 activation. The results have revealed three new and important mechanistic details of the mechanism of action of IPA-3. First, we demonstrate that IPA-3 binds Pak1 covalently, in a time- and temperature-dependent manner. Intriguingly, this interaction is highly selective, saturable and of low stoichiometry although the chemical basis for this selectivity and the precise nature of the adducts are, as yet, unknown. Second, we show that by binding Pak1, IPA-3 prevents binding of the Pak1 activator Cdc42, thus providing a mechanistic explanation for how IPA-3 inhibits Pak1 activation. Thirdly, we demonstrate that IPA-3 binds directly to the Pak1 autoregulatory domain. Inhibition of kinases by selective covalent modification of active site residues is now well established (20–23). The findings with IPA-3 suggest that covalent modification of sites outside of the active site might offer additional opportunities for target inhibition. Indeed, covalent inhibitors of Polo-like kinase have been identified that target the functionally important Polo box domain rather than the kinase domain (24). Other examples of covalent allosteric inhibitors targeting non-kinase enzymes have also been described (25, 26). Kinase inhibitors acting by this mechanism may exhibit greater kinase selectivity and improved pharmacological efficacy (27). Indeed, covalent post-translational modifications occurring outside of active sites such as phosphorylation, ubiquitinylation and SUMOylation are important regulators of enzyme activity in biological systems. This suggests that small-molecule inhibitors for other enzymes might be found that exploit the regulatory power of covalent modification distal to the active site.
Finally, our findings implicate Pak1 in the formation of actin-rich ruffles stimulated by PMA. Pak1 localized to these structures and ruffling was reversibly inhibited by IPA-3. Other proteins previously localized to dorsal membrane ruffles include cortactin and Arp2/3 complex, two known Pak1 substrates (28). Although the biological function of these structures and the signaling pathways that regulate them remain to be determined, they may play a role in of receptor internalization and/or extracellular matrix degradation (29). The results presented here suggest that Pak1 plays a central role in coordinating the underlying cytoskeletal organization.
Grant support - This work was funded by Fondation pour la Recherche Medicale (JV), Department of Defense NF Research Program (W81XWH-05-1-0200), an AACR-FCCC Career Development Award, NIH GM083025, and the Pennsylvania Department of Health (JRP). Additional support was provided by an NCI CORE grant to Fox Chase Cancer Center (CA006927) and an appropriation from the Commonwealth of Pennsylvania.
Pak1 & Cdc42, PRK1 and mini-N-WASP plasmids were generously provided by J. Chernoff, H. Mott and W. Lim, respectively. We thank B. Turk and J. Chernoff for comments on the manuscript and the FCCC Organic Synthesis Facility for chemistry support.
Supplemental Data: Synthesis and characterization of 14C-labeled IPA-3 (2,2′-dihydroxyl-1,1′-dinaphthyldisulfide)