Active mutants of the human p38α MAP kinase

Mitogen Activated Protein (MAP) kinases compose a family of serine/threonine kinases, which function in many signal transduction pathways affecting various cellular phenotypes. Although the abundance of available data, the exact role of each MAP kinase is not completely defined, in part due to the inability to activate MAP kinase molecules individually and specifically. Based on activating mutations found in the yeast MAP kinase p38/Hog1 (Bell et al., 2001, J. Biol. Chem., 276, 25951-8) we designed and constructed single and multiple mutants of human MAP kinase p38α. Single (p38, p38, p38) and subsequent double (p38 ,p38) mutants acquired high intrinsic activity independent of any upstream regulation and reached levels of 10% and 25% respectively, in reference to the dually phosphorylated wild type p38α. The active p38 mutants have retained high specificity toward p38 substrates and were inhibited by the p38’s specific inhibitors SB-203580 and PD-169316. We also show that similar mutations can render p38γ active as well. Based on the available structures of p38 and ERK2, we have analyzed the p38 mutants and identified a hydrophobic core stabilized by three aromatic residues Tyr69, Phe327, and Trp337 in the vicinity of L16 loop region. Upon activation, a segment of the L16 loop, including Phe327, is becoming disordered. Structural and analysis suggest that the active p38 mutants emulate the conformational changes naturally imposed by dual phosphorylation, namely, destabilization of the hydrophobic core. Essentially, the hydrophobic core is an inherent stabilizer that maintains low basal activity level in unphosphorylated p38. 2 by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from