Members of the GCK-VI subfamily of Ste20 kinases regulate a Caenorhabditis elegans ClC anion channel and vertebrate SLC12 cation-chloride cotransporters. With no lysine (K) or WNK protein kinases interact with and activate the mammalian GCK-VI kinases, PASK and OSR-1. We demonstrate here for the first time that GCK-VI kinases play an essential role in whole animal osmoregulation. RNA interference (RNAi) knockdown of the single C. elegans GCK-VI kinase, GCK-3, dramatically inhibits systemic volume recovery and survival following hypertonic shrinkage. Tissue-specific RNAi suggests that GCK-3 functions primarily in the hypodermis and intestine to mediate volume recovery. The single C. elegans WNK kinase, WNK-1, binds to GCK-3 and
wnk-1 knockdown gives rise to a phenotype qualitatively similar to that of
gck-3(RNAi) worms. Knockdown of the two kinases together has no additive effect, suggesting that WNK-1 and GCK-3 function in a common pathway. We postulate that WNK-1 functions upstream of GCK-3 in a manner similar to that postulated for its mammalian homologues. Phylogenetic analysis of kinase functional domains suggests that the interaction between GCK-VI and WNK kinases first occurred in an early metazoan and therefore likely coincided with the need of multicellular animals to tightly regulate transepithelial transport processes that mediate systemic osmotic homeostasis. Key words: cell volume regulation, osmotic stress, osmoregulation.