Abstract
In the structure of autoinhibited EphA2 tyrosine kinase reported herein, we have captured the entire activation segment, revealing a previously unknown role of the conserved Arg762 in kinase autoinhibition by interacting with the essential Mg2+-chelating Asp757. While it is well known that this Arg residue is involved in an electrostatic interaction with the phospho-residue of the activation loop to stabilize the active conformation, our structure determination revealed a new role for the Arg, acting as a switch between the autoinhibited and activated conformations. Mutation of Arg762 to Ala in EphA2 sensitized Mg2+ response, resulting in enhanced kinase catalytic activity and Mg2+ cooperativity. Furthermore, mutation of the corresponding Arg/Lys to Ala in PKA and p38MAPK also exhibited similar behavior. This new salt bridge-mediated switch may thus be an important mechanism of activation on a broader scope for kinases which utilize autophosphorylation.
Highlights
In the structure of autoinhibited EphA2 tyrosine kinase reported we have captured the entire activation segment, revealing a previously unknown role of the conserved Arg[762] in kinase autoinhibition by interacting with the essential Mg2+-chelating Asp[757]
Among all the control mechanisms, the key aspect of regulation of most protein kinases is phosphorylation of one or more residues located in the activation segment[3,4,5]
Mutation of the conserved Arg/Lys to Ala in EphA2, as well as, two other Ser/Thr kinases, PKA and p38MAPK, resulted in enhanced catalytic activity and Mg2+ cooperativity. These results suggest that the conserved Arg/Lys serves as a novel role of regulating the kinase catalytic activity through modulation of the Mg2+ ions binding, which may be an important feature for many protein kinases
Summary
In the structure of autoinhibited EphA2 tyrosine kinase reported we have captured the entire activation segment, revealing a previously unknown role of the conserved Arg[762] in kinase autoinhibition by interacting with the essential Mg2+-chelating Asp[757]. We have determined the unphosphorylated inactive EphA2 structure at 1.9 Å-resolution, which reveals a previous unknown role of Arg[762] (corresponding to Lys[189] in PKA) in the autoinhibition state by interacting with the conserved Mg2+-chelating Asp[757]. Mutation of the conserved Arg/Lys to Ala in EphA2, as well as, two other Ser/Thr kinases, PKA and p38MAPK, resulted in enhanced catalytic activity and Mg2+ cooperativity. These results suggest that the conserved Arg/Lys serves as a novel role of regulating the kinase catalytic activity through modulation of the Mg2+ ions binding, which may be an important feature for many protein kinases
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