Catalysis and function of the p38 alpha.MK2a signaling complex.
The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.
Department of Immunology and Inflammation, Research and Development Center, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Road, Ridgefield, Connecticut 06877, USA.
Susan M
SM
Lukas
Rachel R
RR
Kroe
Jessi
J
Wildeson
Gregory W
GW
Peet
Lee
L
Frego
Walter
W
Davidson
Richard H
RH
Ingraham
Christopher A
CA
Pargellis
Mark E
ME
Labadia
Brian G
BG
Werneburg
D017398:Alternative Splicing; D000818:Animals; D002151:Calorimetry; D002384:Catalysis; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D048308:Mitogen-Activated Protein Kinase 14; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000502:physiology*; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D014461:Ultracentrifugation; D000099190:MAP-Kinase-Activated Kinase 2
D016650:Fluorescein-5-isothiocyanate; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D048308:Mitogen-Activated Protein Kinase 14; D020928:Mitogen-Activated Protein Kinases; D010446:Peptide Fragments; D017346:Protein Serine-Threonine Kinases; D000099190:MAP-Kinase-Activated Kinase 2