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Significance of activation loop phosphorylation in protein kinase A studied by H/D exchange and X‐ray crystallography
Author(s) -
Steichen Jon Martin,
Kuchinskas Michael P,
Iyer Ganesh H,
Li Sheng,
Woods Virgil L,
Taylor Susan S
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.709.9
Subject(s) - autophosphorylation , phosphorylation , kinase , protein kinase a , allosteric regulation , chemistry , enzyme activator , hydrogen–deuterium exchange , enzyme , protein phosphorylation , mapk14 , biochemistry , cyclin dependent kinase 2 , mass spectrometry , chromatography
Protein phosphorylation is possibly the most important mechanism for regulation in eukaryotic cells. Over 500 protein kinases are encoded by the human genome and the catalytic subunit of cAMP‐dependent protein kinase (PKA) serves as a prototype for the entire family of protein kinases. The activation loop is a critical motif for all protein kinases and in many kinases, such as PKA, the activation loop must be phosphorylated in order for the kinase to be active. The inactive form of PKA can be purified by mutating an arginine to alanine (R194A) in the activation loop which prevents autophosphorylation of T197, the critical phosphorylation site. The unphosphorylated enzyme is less active and less stable based on unfolding and kinetic studies. Hydrogen/deuterium exchange coupled to mass spectrometry was used to compare the inactive and active states of PKA. Much of the enzyme is more solvent exposed in the inactive state including regions distant from the activation loop, demonstrating that phosphorylation induces large allosteric changes in the enzyme. Additionally, diffracting crystals of the R194A mutant have been grown and these crystals will provide a structural comparison of the phosphorylated and unphosphorylated states of PKA. This research was supported by NIH Grant GM19301.