Abstract 32: Investigating the Autoactivation of p38α Mitogen-Activated Protein Kinase Mediated by Transforming Growth Factor-β-Activated Protein Kinase Binding Protein 1
Activation of p38α MAPK (p38α), by phosphorylation of two residues in the TGY motif of the activation loop, can occur independently of upstream kinases. One such mechanism involves the scaffolding protein Transforming Growth Factor-β-activated protein kinase binding protein 1 (TAB1). Under certain circumstances, such as myocardial ischemia, this activation can aggravate lethal injury. It is one of a few examples of activating autophosphorylation and poses a conundrum. How does an inactive kinase, and therefore with low affinity for ATP, phosphorylate its own activation loop when ATP binding is a prerequisite step for phosphotransfer? The aim of this study was to characterize the TAB1 binding of p38α. The binding characteristics of p38α and TAB1 were determined by Isothermal Titration Calorimetry, followed by the binding of p38α and ATPγS, a slowly hydrolysable form of ATP, in the presence and absence of TAB1. The binding of TAB1 to p38α increased significantly the affinity of p38α for ATP. Following the identification of a key region in TAB1 responsible for p38α binding, a synthetic peptide encompassing this region was used to analyze the biophysical and biological consequences of TAB1 binding. In vitro kinase assays were used to test the biochemical characteristics using a combination of wildtype kinase, kinase dead (K53M) or both in the absence or presence of TAB1(371-416). Using an antibody specific to the dual phosphorylation of the TGY motif as a readout, TAB1 binding to p38α increased p38α autophosphorylation in cis. NMR was employed to map the interaction surfaces between of p38α and TAB1 and to analyze the effect of TAB1-binding on p38α. The residues identified as important for the interaction between TAB1 and p38α were mutated and tested in cell free and biological systems to confirm their role as critical determinants for binding. In conclusion, we have further elucidated a mechanism whereby TAB1 binding to p38α alters the conformation of p38α, increasing its affinity for ATP and thereby facilitating autophosphorylation. We have identified the binding contacts of TAB1 and p38α that may be important in the design of therapeutics enabling selective and circumstance-specific inhibition of p38α activation.
- © 2012 by American Heart Association, Inc.