Dynamic activation and regulation of the mitogen-activated protein kinase p38

Abstract

Mitogen-activated protein kinases, which include p38, are essential for cell differentiation and autophagy. The current model for p38 activation involves activation-loop phosphorylation with subsequent substrate binding leading to substrate phosphorylation. Despite extensive efforts, the molecular mechanism of activation remains unclear. Here, using NMR spectroscopy, we show how the modulation of protein dynamics across timescales activates p38. We find that activation-loop phosphorylation does not change the average conformation of p38; rather it quenches the loop ps-ns dynamics. We then show that substrate binding to nonphosphorylated and phosphorylated p38 results in uniform µs-ms backbone dynamics at catalytically essential regions and across the entire molecule, respectively. Together, these results show that phosphorylation and substrate binding flatten the energy landscape of the protein, making essential elements of allostery and activation dynamically accessible. The high degree of structural conservation among ser/thr kinases suggests that elements of this mechanism may be conserved across the kinase family.

Keywords: MAP kinase; NMR dynamics; NMR spectroscopy; kinase activation; signaling.

Fig. 1.

Phosphorylation affects fast timescale dynamics at the activation loop. (A) p38 adopts a typical kinase fold with an N- and a C-terminal lobe. Gly-rich loop, KIM-binding site, hinge, activation loop, and the MAPK-specific insert are highlighted. (B–F) Cartoon representation of the p38 states investigated in this work: (B) np-p38, (C) dp-p38, (D) np-p38⋅MKK3b_KIM, (E) dp-p38⋅MKK3b_KIM, and (F) np-p38⋅MKK3b_KIM⋅AMP-PNP. Activation loop (green), KIM-peptide (dark blue), and AMP-Mg⁺²–binding sites (sticks) are highlighted. (G) Chemical-shift perturbations (CSP) upon activation-loop phosphorylation vs. residue number. (H) The ¹⁵N longitudinal relaxation rates (R₁) and (I) transverse relaxation rates (R₂) for np-p38 (black) and dp-p38 (red) vs. residue number. The Gly-rich loop (blue), hinge (cyan), and activation loop (green) are highlighted.

Fig. 2.

KIM binding induces exchange dynamics beyond the peptide-binding site. Clusters of residues with uniform exchange dynamics (rates and population), color coded on the basis of k_ex, are shown for (A) np-p38 (magenta, >2,500 s⁻¹; cyan, >2,500 s⁻¹; orange, 1,687 ± 134 s⁻¹; and green, 1,452 ± 187 s⁻¹) and (B) np-p38⋅MKK3b_KIM (magenta, >2,500 s⁻¹; cyan, 1,251 ± 68 s⁻¹; and orange, 2,403 ± 332 s⁻¹) (blue sticks/surface). Residues that cannot be fit within a cluster in a statistically meaningful manner are shown as black spheres. Number of residues in the largest clusters is shown. (C) MKK3b_KIM peptide binding (blue sticks/surface) leads to CSPs (beige) only in the KIM-binding pocket, whereas residues beyond the KIM-binding pocket cluster with uniform exchange dynamics (magenta spheres; includes the KIM-binding pocket, the hinge, the N-terminal lobe, and the activation loop). (D) Residues with µs-ms dynamics are highlighted (magenta) on the histogram showing the ¹H/¹⁵N CSPs of np-p38 upon MKK3b_KIM binding vs. residue number.

Fig. 3.

Phosphorylation and KIM-peptide binding induces uniform exchange dynamics throughout p38. Clusters with uniform exchange dynamics, residues color coded on the basis of on k_ex, in (A) dp-p38 (magenta, 1,304 ± 58 s⁻¹; cyan, 1,649 ± 106 s⁻¹; orange, 962 ± 98 s⁻¹; and green, >2,500 s⁻¹) and (B) dp-p38⋅MKK3b_KIM (magenta, 1,826 ± 80 s⁻¹). Residues that could not be fit within a cluster in a statically meaningful manner are shown as black spheres. (C) Representative ¹⁵N ct-CPMG dispersion data (dp-p38⋅MKK3b_KIM) for residues Thr106 (cyan), Asn159 (green), Leu246 (orange), and Phe274 (blue); corresponding residues are highlighted in B. The lines show cluster-fits to the Richard–Carver equation, and error bars are derived from duplicate measurements.

Fig. 4.

Dynamic activation of p38. KIM-peptide binding (blue sticks/surface) leads to uniform µs-ms dynamics between the hinge and activation loop, while the activation loop stays dynamic in the ps-ns timescale (yellow arrow). Phosphorylation (green balls) rigidifies the activation loop and, together with KIM-peptide binding, manipulates the motions that regulate the energy landscape of p38.