Mechanistic studies of the autoactivation of PAK2: a two-step model of cis initiation followed by trans amplification

Abstract

Protein kinase activation, via autophosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation-dependent signaling cascades. Despite the prevalence of this reaction and its importance in biological regulation, the molecular mechanisms of autophosphorylation are poorly understood. In this study, we developed a kinetic approach to distinguish quantitatively between cis- and trans-pathways in an autocatalytic reaction. Using this method, we have undertaken a detailed kinetic analysis for the autoactivation mechanism of p21-activated protein kinase 2 (PAK2). PAK2 is regulated in vivo and in vitro by small GTP-binding proteins, Cdc42 and Rac. Full activation of PAK2 requires autophosphorylation of the conserved threonine, Thr(402), in the activation loop of its catalytic kinase domain. Analyses of the time courses of substrate reaction during PAK2 autoactivation suggest that autophosphorylation of Thr(402) in PAK2 obeys a two-step mechanism of cis initiation, followed by trans amplification. The unphosphorylated PAK2 undergoes an intramolecular (cis) autophosphorylation on Thr(402) to produce phosphorylated PAK2, and this newly formed active PAK2 then phosphorylates other PAK2 molecules at Thr(402) in an intermolecular (trans) manner. Based on the kinetic equation derived, all microscopic kinetic constants for the cis and trans autophosphorylation have been estimated quantitatively. The advantage of the new method is not only its usefulness in the study of fast activation reactions, but its convenience in the study of substrate effects on modification reaction. It would be particularly useful when the regulatory mechanism of the autophosphorylation reaction toward certain enzymes is being assessed.

FIGURE 1.

Western blot analysis of PAK2. Lane 1, inactive GST-PAK2. Lane 2, self-activated GST-PAK2 (in the presence of GST-Cdc42L61). Phosphorylation at Ser¹⁴¹, Ser^192/197, and Thr⁴⁰² in PAK2 was monitored with the phosphospecific antibodies (pAb): pPAK2(Ser¹⁴¹) Ab, pPAK2(Ser^192/197) Ab, and pPAK2(Thr⁴⁰²) Ab, respectively. Total PAK2 was analyzed by Coomassie Blue staining.

FIGURE 2.

Effect of interaction between Cdc42L61 and PAK2 on PAK2-catalyzed reaction. A, time courses of PAK2-catalyzed reactions. The reaction mixture contained standard assay buffer mixture and 200 μm MLCtide. The reactions were initiated by adding 3 nm phosphorylated PAK2 (trace 1) and 7.5 nm unphosphorylated PAK2 (trace 2). No significant enzyme activity was observed upon addition of unphosphorylated PAK2 in the absence of Cdc42L61. Following addition of 3 nm phosporylated PAK2 (as the arrow indicated), a linear increase in absorbance at 340 nm occurred (trace 3). B, dependence of the initial rate of the PAK2-catalyzed reaction on Cdc42L61 concentration. The reaction mixture contained standard assay buffer mixture, 200 μm MLCtide and different concentrations of Cdc42L61. The reaction was initiated by addition of 3 nm phosphorylated PAK2 into the assay system.

SCHEME 3

FIGURE 3.

Time courses of substrate reaction in the presence of different concentrations of the full-length PAK2. The concentrations of Cdc42L61 and MLCtide in the assay system were 0.8 μm and 200 μm respectively. The full-length PAK2 was added to the reaction mixture to start the reaction. Final concentrations of PAK2 were 7.5, 11.25, 15, and 22.5 nm for curves 1–4, respectively. Other conditions were the same as in Fig. 2. The data were fitted to Equation 1 to determine the kinetic parameters, v_s, k_obs, and β. The solid black lines are the best fitting results according to Equation 1. Inset: plots of the observed rate constant, k_obs, against enzyme concentration, [T]₀.

FIGURE 4.

Time courses of substrate reaction in the presence of different concentrations of Cdc42L61. The reaction was started by the addition of the full-length PAK2 to the reaction mixture. The final concentration of enzyme and MLCtide was 7.5 nm and 200 μm, respectively. Concentrations of Cdc42L61 were 1.6, 3.2, 4.8, 8, and 12.6 μm for curves 1–5, respectively. Other conditions were the same as in Fig. 2. The data were fitted to Equation 1 to determine the kinetic parameters, v_s, k_obs, and β. Inset: plot of the observed rate constant, k_obs against Cdc42L61 concentration, [L]. The solid line represents the best fitting result according to k_obs = (A[T₀] + k_e)[L]/(K_L + [L]) with K_L = 6.3 μm and A[T]₀ + k_e = 0.066 s⁻¹.

FIGURE 5.

Time courses of substrate reaction in the presence of different concentrations of MLCtide. The reaction was started by the addition of PAK2 to the reaction mixture. The final concentration of enzyme and Cdc42L61 ware 7.5 nm and 1.6 μm, respectively. Concentrations of MLCtide were 100, 200, 300, and 400 μm for curves 1–4, respectively. Other conditions were the same as in Fig. 2. The data were fitted to Equation 8 to determine the kinetic parameters, v_s, A, and k_e.

FIGURE 6.

Effect of MLCtide concentration on PAK2 autoactivation. A, plot of the steady-state velocities v_s against substrate concentration, [S]. The solid line represents the best fitting result according to Michealis-Menten equation with K_S = 292 μm, k₂ = 34 s⁻¹. B, plot of the apparent second-order rate constant A against substrate concentration, [S]. The continuous line represents the best fitting result according to Equation 5 with k₊₀ = 14.4 μm⁻¹ s⁻¹, and a fixed K_S = 292 μm. C, plot of the reciprocal of the apparent second-order rate constant, A, against substrate concentration, [S]. D, plot of the intra-initiation rate constant k_e against substrate concentration, [S]. The solid line represents the best fitting result with k_e of 0.0254 ± 0.0007 s⁻¹.