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Functional insights into the activation mechanism of Ste20‐ related kinases
Author(s) -
Gag Kenneth B,
Rios Kerri,
Delpire Eric
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.694.5
Subject(s) - kinase , xenopus , microbiology and biotechnology , strongylocentrotus purpuratus , biology , protein kinase a , heterologous , protein kinase domain , gene , genetics , sea urchin , mutant
Heterologous expression of sea urchin ( Strongylocentrotus purpuratus ) OSR1 (suOSR1) cRNA with mouse WNK4 cRNA and mouse NKCC1 cRNA in Xenopus laevis oocytes activated the cotransporter indicating evolutionary conservation of the WNK4‐ OSR1‐NKCC signaling pathway. However, expression of a suOSR1 kinase mutated to confer constitutive activity did not result in stimulation of the cotransporter. Using a chimeric strategy, we determined that both the mutated catalytic and regulatory domains of the suOSR1 kinase were functional, suggesting that the tertiary structure of full‐length mutated suOSR1 must somehow adopt an inactive conformation. In order to identify the regions or residues which lock the suOSR1 kinase in an inactive conformation, we created and tested several additional chimeras. Coexpression of these chimeras identified several regions in both the catalytic and regulatory domain of suOSR1 which prevented the kinase from acquiring an active conformation. Interestingly, non‐functional suOSR1 chimeras were able to activate mouse NKCC1 when a mouse scaffolding protein, Cab39, was co‐expressed. Chimeras and kinase stabilization with mouse Cab39 has provided some novel insights into the activation mechanism of the Ste20‐related kinases.