Abstract
During apoptotic stress, protein kinase Pak2 is cleaved by caspase 3 to form a heterotetramer that is constitutively activated following autophosphorylation. The active protein kinase migrates slightly slower than the inactive holoenzyme when analyzed by gel filtration, suggesting an expanded conformation. Activation of Pak2 comprises a series of structural changes resulting from caspase cleavage, ATP binding, and autophosphorylation of Pak2. Changes at each step were individually analyzed by amide hydrogen/deuterium exchange coupled with mass spectrometry and compared with inactive Pak2. The auto-inhibited form was shown to bind ATP in the active site, with minor changes in the glycine loop and the autoinhibitory domain (AID). Caspase cleavage produced significant changes in solvent accessibility in the AID and upper lobe of the catalytic domain. Cleavage of ATP-bound Pak2 relaxes the allosteric inhibition, as shown by increased solvent accessibility in the upper and lower lobes, including the G-helix, facilitating the autophosphorylation of two sites required for activation, Ser-141 in the regulatory domain and Thr-402 in the catalytic domain. Autophosphorylation increased the amide hydrogen/deuterium exchange solvent accessibility of the contact region between the AID and the G-helix, the E-F loop, and the N terminus. Thus, activation of Pak2 via caspase cleavage is associated with structural relaxation of Pak2 that allows for complete auto-phosphorylation, resulting in a more comprehensive solvent-exposed and conformationally dynamic enzyme.
Highlights
In the active site there was a net gain of about one deuteron in the 295–309 fragment following caspase cleavage, but not in the 297–309 or 283–294 fragments, suggesting the change in amide H/D exchange was confined to either residue 295 or 296 adjacent to Glu-294 in the C helix, which is an important link to the glycine-rich loop in ATP binding
Pak2 is constitutively activated by caspase 3 during apoptosis in response to apoptotic stresses such as UV radiation and other interact with the G helix of the catalytic domain (Fig. 1)
There was no sequence coverage of the G helix, frag- ture of caspase-cleaved Pak2 by gel filtration shows that the ments that include the loop preceding (436 – 451 and 437– 451) caspase cleavage products of Pak2 remain associated as a hetand following [455– 473] the G helix, but not the H helix
Summary
Materials—Bovine thrombin was obtained from Amersham Pharmacia Biotech. Pepsin immobilized on 6% agarose beads was purchased from Pierce. Autophosphorylation of cleaved Pak was carried out at a final concentration of 10 mg/ml Pak in a 20-l reaction mixture containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 15 mM MgCl2, 2 mM dithiothreitol, and 10 mM ATP, and incubated at 34 °C for 30 min. All fractions containing peptides were subjected to tandem mass spectrometry (Q-TOF UltimaGlobal and QSTAR XL oMALDI MS/MS, Applied Biosystems) to identify the fragments, based on the primary sequences using Protein Prospector [28]. The time between thawing of the sample and spectrum retrieval was ϳ3 min. Each spectrum was calibrated based on the flight times of two sequenced peaks whose calculated undeuterated m/z were 923.45 and 1697.84 Both peaks had high intensity even after the sample was deuterated. The Ds(t) is the amount of residual deuterium on the side chains at time t
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