Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK

Clinton A. Taylor, Yu Chi Juang, Svetlana Earnest, Samarpita Sengupta, Elizabeth J. Goldsmith, Melanie H. Cobb

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The related protein kinases SPAK and OSR1 regulate ion homeostasis in part by phosphorylating cation cotransporter family members. The structure of the kinase domain of OSR1 was determined in the unphosphorylated inactive form and, like some other Ste20 kinases, exhibited a domain-swapped activation loop. To further probe the role of domain swapping in SPAK and OSR1, we have determined the crystal structures of SPAK 63-403 at 3.1 Å and SPAK 63-390 T243D at 2.5 Å resolution. These structures encompass the kinase domain and different portions of the C-terminal tail, the longer without and the shorter with an activating T243D point mutation. The structure of the T243D protein reveals significant conformational differences relative to unphosphorylated SPAK and OSR1 but also has some features of an inactive kinase. Both structures are domain-swapped dimers. Sequences involved in domain swapping were identified and mutated to create a SPAK monomeric mutant with kinase activity, indicating that monomeric forms are active. The monomeric mutant is activated by WNK1 but has reduced activity toward its substrate NKCC2, suggesting regulatory roles for domain swapping. The structure of partially active SPAK T243D is consistent with a multistage activation process in which phosphorylation induces a SPAK conformation that requires further remodeling to build the active structure.

Original languageEnglish (US)
Pages (from-to)5063-5071
Number of pages9
JournalBiochemistry
Volume54
Issue number32
DOIs
StatePublished - Jul 24 2015

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Protein Kinases
Phosphotransferases
Switches
Chemical activation
Phosphorylation
Point Mutation
Dimers
Conformations
Cations
Homeostasis
Crystal structure
Ions
Substrates
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK. / Taylor, Clinton A.; Juang, Yu Chi; Earnest, Svetlana; Sengupta, Samarpita; Goldsmith, Elizabeth J.; Cobb, Melanie H.

In: Biochemistry, Vol. 54, No. 32, 24.07.2015, p. 5063-5071.

Research output: Contribution to journalArticle

Taylor, Clinton A. ; Juang, Yu Chi ; Earnest, Svetlana ; Sengupta, Samarpita ; Goldsmith, Elizabeth J. ; Cobb, Melanie H. / Domain-Swapping Switch Point in Ste20 Protein Kinase SPAK. In: Biochemistry. 2015 ; Vol. 54, No. 32. pp. 5063-5071.
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