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Phospho-Priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation
- Source :
- Biochemistry. 56:5112-5124
- Publication Year :
- 2017
- Publisher :
- American Chemical Society (ACS), 2017.
-
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.
- 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
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 56
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....dfa865fe8d234a59948828f0ac7534d8