Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Protein kinases SPAK (STE20/SPS1-related proline-rich kinase) and OSR1 (oxidative stress responsive 1) regulate ion homeostasis.
• They achieve this by phosphorylating cation cotransporter family members.
• Previous studies showed OSR1's inactive form has a domain-swapped activation loop.

Purpose of the Study:

• To investigate the role of domain swapping in SPAK and OSR1.
• To determine the structural basis of SPAK activation and regulation.

Main Methods:

• X-ray crystallography was used to determine the structures of SPAK (63-403) and a T243D mutant (63-390).
• Mutagenesis was employed to create a monomeric SPAK mutant.
• Kinase activity assays were performed on wild-type and mutant SPAK proteins.

Main Results:

• Both determined SPAK structures revealed domain-swapped dimers, similar to OSR1.
• A monomeric SPAK mutant was generated and shown to possess kinase activity.
• The T243D mutant exhibited conformational differences, suggesting a multistage activation process.

Conclusions:

• Monomeric forms of SPAK are catalytically active.
• Domain swapping in SPAK and OSR1 appears to play a regulatory role in their function.
• The activation of SPAK likely involves a multistage process initiated by phosphorylation.