IPA-3 binds the regulatory domain of Pak1
IPA-3 () 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 (). 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 [32
P]-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 (; 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.
IPA-3 binding to the RD inhibits Cdc42 binding
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 (; 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.
IPA-3 inhibits Pak1-Cdc42 binding
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 (). Treatment with IPA-3 inhibited the binding of Pak1 to Cdc42 (compare lanes 2 and 3), supporting the in vitro results.
IPA-3 binds Pak1 covalently
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 ). 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 (, 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 ().
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 (, 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 [14
C]-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 (). Phosphor imager analysis of the proteolytic digests demonstrated ~ 37% of the Pak1-bound IPA-3 was associated with the stable catalytic domain (, 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 (, 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.
IPA-3 binding to Pak1 is selective
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 (, 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 (). 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 (). 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.
IPA-3 binding and inhibitory activity is reversed by reducing environments in vitro and in vivo
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 ().
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
) 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 (). 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 (). 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 (). We therefore conclude that the effect of IPA-3 is reversible in live cells, most likely through reductive release of IPA-3.
IPA-3 reversibly inhibits PMA-induced membrane ruffling in cells