1. Phospho-Priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation
- Author
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Jui-Hung Weng, Wen-Jin Wu, Wei-Cheng Huang, Yoshiaki Kawano, Ming-Daw Tsai, Kai-Fa Huang, Liang-Hin Lim, Yoshitaka Bessho, Eric S.-W. Chen, Yu-Hou Chen, Shun-Chang Wang, Xiao-Xia Liu, Tsutomu Matsui, and Jiahn-Haur Liao
- Subjects
Models, Molecular ,Threonine ,0301 basic medicine ,Saccharomyces cerevisiae Proteins ,Cell Cycle Proteins ,Protein Serine-Threonine Kinases ,Biology ,Biochemistry ,AKT3 ,MAP2K7 ,03 medical and health sciences ,Protein Domains ,Scattering, Small Angle ,Serine ,c-Raf ,Phosphorylation ,Nuclear Magnetic Resonance, Biomolecular ,MAPK14 ,Serine/threonine-specific protein kinase ,Protein-Serine-Threonine Kinases ,Autophosphorylation ,Checkpoint Kinase 2 ,Phosphothreonine ,030104 developmental biology ,Protein kinase domain ,Protein Multimerization ,DNA Damage - Abstract
The vast majority of in vitro structural and functional studies of the activation mechanism of protein kinases use the kinase domain alone. Well-demonstrated effects of regulatory domains or allosteric factors are scarce for serine/threonine kinases. Here we use a site-specifically phosphorylated SCD1-FHA1-kinase three-domain construct of the serine/threonine kinase Rad53 to show the effect of phospho-priming, an in vivo regulatory mechanism, on the autophosphorylation intermediate and specificity. Unphosphorylated Rad53 is a flexible monomer in solution but is captured in an asymmetric enzyme:substrate complex in crystal with the two FHA domains separated from each other. Phospho-priming induces formation of a stable dimer via intermolecular pT-FHA binding in solution. Importantly, autophosphorylation of unprimed and phospho-primed Rad53 produced predominantly inactive pS350-Rad53 and active pT354-Rad53, respectively. The latter mechanism was also demonstrated in vivo. Our results show that, while Rad53 can display active conformations under various conditions, simulation of in vivo regulatory conditions confers functionally relevant autophosphorylation.
- Published
- 2017