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Abstract
<p class="first" id="P3">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
solved 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/OSR1, we have determined the crystal structures of SPAK 63–403 at 3.1 Å and SPAK
63–390 T243D at 2.5 Å resolutions. These structures encompass the kinase domain and
different portions of the C-terminal tail, the longer without, and the shorter with
an activating point mutation T243D. 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 the partially active
SPAK T243D is consistent with a multi-stage activation process in which phosphorylation
induces a SPAK conformation that requires further remodeling to build the active structure.
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