ON THE SUBSTRATE RECOGNITION AND AUTOINHIBITORY DOMAIN OF SPAK

Abstract

Threonines targeted by SPAK for phosphorylation have been identified in NKCC1, NKCC2, and NCC. However, what constitutes the substrate recognition of the kinase is still unknown. Using site‐directed mutagenesis and functional measurement of NKCC1 activity in Xenopus laevis oocytes, we determined that SPAK recognizes two threonine residues separated by four amino acids. Addition or removal of a single residue abrogated SPAK activation of NKCC1. Although both threonines are followed by hydrophobic residues, only the first residue is essential for SPAK activation of the cotransporter. Negatively charged residues around the sites targeted for phosphorylation by other kinases have been shown to be involved in substrate recognition. Thus, we mutated two aspartate residues flanking the two threonines. Whereas alanine mutation of the upstream residue had no effect, alanine substitution of the downstream residue inhibited cotransporter activity. However, conserved substitution of this residue into glutamate preserved NKCC1 function. SPAK activity requires phosphorylation of two residues by WNK kinases. We found that mutating T243E combined with S383D rendered the kinase constitutively active. Surprisingly, S383A mutation or mutation of residues surrounding this residue also resulted in an active kinase. These data indicate the possibility of an inhibitory domain which is affected by WNK phosphorylation.