The Na-K-2Cl cotransporter, NKCC1, is expressed in a variety of cells where it plays a multitude of functions. Numerous studies have demonstrated that NKCC1 is a phosphoprotein and shown that stimuli resulting in an increase of Na-K-2Cl cotransport activity also cause an increase in phosphorylation of the cotransport protein. In 2002, we reported a protein-protein interaction between Ste20p-related Proline Alanine-rich Kinase (SPAK) and NKCC1 through two putative binding motifs (R/K)Fx(V/I) located within in the cytosolic amino-terminal portion of NKCC1. Here, we demonstrate the necessity for at least one SPAK binding motif in the amino terminus of NKCC1. We also confirm that the conserved phenylalanine residue within the motif is critical for SPAK binding and phosphorylation of the cotransporter. Furthermore, we correlate the in vivo activation of NKCC1 with the in vitro phosphorylation studies using recombinant SPAK fusion proteins. We also examine the role of AATYK1, a putative tyrosine kinase interacting with SPAK. We show that AATYK1markedly inhibits cotransporter activity, independently of kinase activity. Mutagenesis of the two SPAK-binding motifs in AATYK1 completely abrogated this effect. We also identified a PP1 docking motif in AATYK1 and demonstrated that mutation of this motif also prevented inhibition of NKCC1 activity. Taken together, our data are consistent with AATYK1 indirectly inhibiting the SPAK activation of the cotransporter by scaffolding an inhibitory phosphatase in proximity to a stimulatory kinase. This work was supported by NIH grants NS36758 and GM074771.