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. 2001 Aug;21(15):5179-89.
doi: 10.1128/MCB.21.15.5179-5189.2001.

Conformational switch and role of phosphorylation in PAK activation

G Buchwald  1 , E Hostinova, M G Rudolph, A Kraemer, A Sickmann, H E Meyer, K Scheffzek, A Wittinghofer
Affiliations

Conformational switch and role of phosphorylation in PAK activation

G Buchwald et al. Mol Cell Biol. 2001 Aug.

Abstract

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.

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Figures

FIG. 1
FIG. 1
Structural model of αPAK, using as a guideline the X-ray structure of a complex between a regulatory fragment from residues 70 to 175 and the complete CD (residues 249 to 545) from human αPAK (18). The regulatory part of the molecule is in dark blue structural elements not included in the three-dimensional structure are in light blue. Approximate locations of residues relevant to the results presented in this report are indicated. It was previously assumed that inhibition by the inhibitory switch (IS) domain and dimerization via the Di motif is relieved by binding of Cdc42 to the CRIB region. KI, kinase inhibitory linker.
FIG. 2
FIG. 2
Intrinsic and Cdc42-stimulated protein kinase activity of αPAK. αPAK (1.7 μM) was incubated in kinase buffer in the presence of 3.3 μM [γ-32P]ATP and 54 μM MBP, in the absence and presence of Cdc42, in the GDP- or GppNHp-bound conformation. The reaction mixture was incubated at 37°C, and samples from the indicated time points were analyzed for phosphorylation in arbitrary units as described in Materials and Methods.
FIG. 3
FIG. 3
Equilibrium measurement of the PAK-Cdc42 interaction. The fluorescence emission spectrum of 0.2 μM Cdc42-mGppNHp in buffer C (upper trace) decreases upon treatment with (from top to bottom) 0.5, 1.0, 1.5, 2.0, and 2.5 μM αPAK. (B) Cdc42-mGppNHp (0.2 μM) was treated with increasing amounts of αPAK as indicated, and the decrease in fluorescence was fitted to a binding equation with 1:1 stoichiometry.
FIG. 4
FIG. 4
Proteolytic digestion of αPAK. (A) αPAK (66 μM) was digested with chymotrypsin (1,000:1; wt/wt). At the indicated time intervals, aliquots of the reaction mixture were treated with protease inhibitors and analyzed by SDS-PAGE as described in Materials and Methods. (B) The reaction mixture from chymotrypsin digestion was applied to a Superdex 75 (10/30) gel filtration column equilibrated in buffer B, and the eluted fractions were analyzed by SDS-PAGE.
FIG. 5
FIG. 5
Proteolyzed PAK is catalytically active. (A) Autoradiogram showing the results of protein kinase assay of the RD-CD complex without Cdc42, with either Cdc42-GDP or Cdc42-GppNHP, using conditions as described in the legend to Fig. 2 and Materials and Methods. Lane 1, kinase assay with FL αPAK in the presence of Cdc42-GppNHp; lane 2, the same reaction in the absence of αPAK. Note that the CD is phosphorylated, whereas RD phosphorylation cannot be detected in the background on MBP. (B) Time course of MBP phosphorylation in (arbitrary units) by the RD-CD complex in the presence or absence of Cdc42-GppNHp. (C) Control incubation with FL αPAK. MBP phosphorylation and autophosphorylation are observed only in the presence of Cdc42-GppNHp.
FIG. 5
FIG. 5
Proteolyzed PAK is catalytically active. (A) Autoradiogram showing the results of protein kinase assay of the RD-CD complex without Cdc42, with either Cdc42-GDP or Cdc42-GppNHP, using conditions as described in the legend to Fig. 2 and Materials and Methods. Lane 1, kinase assay with FL αPAK in the presence of Cdc42-GppNHp; lane 2, the same reaction in the absence of αPAK. Note that the CD is phosphorylated, whereas RD phosphorylation cannot be detected in the background on MBP. (B) Time course of MBP phosphorylation in (arbitrary units) by the RD-CD complex in the presence or absence of Cdc42-GppNHp. (C) Control incubation with FL αPAK. MBP phosphorylation and autophosphorylation are observed only in the presence of Cdc42-GppNHp.
FIG. 6
FIG. 6
Dimerization of PAK and the RD-CD complex in the presence of Cdc42 and its affinity to Cdc42. (A) Elution profile of the isolated RD fragment and its complex with Cdc42-GppNHp on a gel filtration (Superdex 75 (26/60)) column with buffer C, which elute with the indicated volumes, plotted against the log of the molecular mass on a calibration curve (B) obtained from marker proteins. The column was equilibrated with a standard where reference proteins 1 to 11 have molecular masses of 81, 67, 43, 35, 29, 25, 17.6, 13.7, 12, 7 and 6.5 kDa, respectively. Calculated elution volume for a Cdc42-RD monomer or dimer complex is indicated. (C) Elution profiles with buffer C on a Superdex 200 (10/30) column of αPAK alone and complexed with Cdc42-GppNHp before and after incubation with 5 mM ATP. The affinity of the RD-CD complex (D) is determined by fluorescence titration with Cdc42-mGppNHp as shown in Fig. 3, giving the indicated affinity of 0.16 μM for the RD-CD complex.
FIG. 7
FIG. 7
Kinetics of interaction between RD and Cdc42. Increasing concentrations of RD were mixed with Cdc42-mGppNHp in a stopped-flow apparatus, and the fluorescence change due to complex formation was recorded. The primary fluorescence transients, of which panel A is an example, were fitted to a single exponential to give kobs. The latter was plotted against the concentration of RD (B) to give the kass, 1.2 × 106 M−1 s−1. The kdiss of 0.02 s−1 was obtained by mixing a preformed RD-Cdc42-mGppNHp complex with excess unlabeled Cdc42-GppNHp as described in Materials and Methods.
FIG. 8
FIG. 8
Autophosphorylation but not Cdc42-GppNHp interaction dissociates the RD-CD complex. (A) GSH-beads were loaded with 30 μg of GST-Cdc42-GppNHp and incubated with 20 μg of the RD-CD complex. The beads were incubated for 30 min at 4°C with buffer (control) or with 1 mM ATP or AppNHp in the presence of excess Mg2+ as indicated, and washed twice with buffer C (wash 1 and 2), and then washed with SDS sample buffer to remove all bound proteins (fraction bound). The starting materials (lanes 1 to 3) and the contents of the washes were analyzed by SDS-PAGE as indicated, together with a molecular weight marker (M). (B) The RD-CD complex in buffer C was incubated with 1 mM ATP, and the affinity to Cdc42-mGppNHp was determined as in Fig. 3. (C) Pull-down assay as in panel A, but with the RD-CD fragment from kinase inactive (K298A) mutant of αPAK. Dots indicate unidentified impurities.
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