Phospho-Priming Confers Functionally Relevant Specificities for Rad53 Kinase Autophosphorylation

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Protein kinase activation studies often use isolated kinase domains, neglecting regulatory domains.
• The impact of regulatory domains and allosteric factors on serine/threonine kinases is understudied.
• Rad53 is a crucial serine/threonine kinase involved in DNA damage response.

Purpose of the Study:

• To investigate the effect of phospho-priming on Rad53 kinase autophosphorylation and specificity.
• To elucidate the structural and functional consequences of phospho-priming in Rad53.
• To compare Rad53 activation under in vitro and simulated in vivo conditions.

Main Methods:

• Utilized a site-specifically phosphorylated three-domain construct (SCD1-FHA1-kinase) of Rad53.
• Employed structural studies (crystallography) and solution-state biophysical techniques.
• Investigated autophosphorylation patterns of unprimed and phospho-primed Rad53.

Main Results:

• Unphosphorylated Rad53 exists as a flexible monomer, while phospho-priming induces a stable dimer via intermolecular pT-FHA binding.
• Autophosphorylation of unprimed Rad53 yielded inactive pS350-Rad53.
• Phospho-primed Rad53 predominantly produced active pT354-Rad53, a mechanism confirmed in vivo.

Conclusions:

• Phospho-priming is a critical in vivo regulatory mechanism for Rad53.
• Simulating in vivo regulatory conditions reveals functionally relevant autophosphorylation.
• Rad53's activity and specificity are significantly influenced by its regulatory domains and phosphorylation state.