On the substrate recognition and negative regulation of SPAK, a kinase modulating Na+-K+-2Cl- cotransport activity

Am J Physiol Cell Physiol. 2010 Sep;299(3):C614-20. doi: 10.1152/ajpcell.00074.2010. Epub 2010 May 12.


Threonines targeted by Ste20-related proline-alanine-rich kinase (SPAK) for phosphorylation have been identified in Na+-K+-2Cl(-) cotransporter type 1 (NKCC1), NKCC2, and Na+-Cl(-) cotransporter (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, in vivo experiments have determined that SPAK activation of the cotransporter only requires a hydrophobic residue after the first threonine. Interestingly, downstream of the second threonine residue, we have identified a conserved aspartic acid residue which is critical for NKCC1 function. Mouse SPAK activity requires phosphorylation of two specific residues by WNK [with no lysine (K)] kinases: a threonine (T243) in the catalytic domain and a serine (S383) in the regulatory domain. We found that mutating the threonine residue into a glutamic acid (T243E) combined with mutation of the serine into an aspartic acid (S383D) rendered SPAK constitutively active. Surprisingly, alanine substitution of S383 or mutation of residues surrounding this residue also resulted in a constitutively active kinase. Interestingly, deletion of amino acids 356-398 identified another serine residue in the catalytic domain (S321) as another putative target of WNK phosphorylation. We found that WNK4 is capable of stimulating the deletion mutant when S321 is present, but not when S321 is mutated into an alanine.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Female
  • Hydrophobic and Hydrophilic Interactions
  • In Vitro Techniques
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Oocytes / metabolism
  • Phosphorylation
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / biosynthesis
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Protein-Serine-Threonine Kinases / physiology*
  • Sodium-Potassium-Chloride Symporters / metabolism*
  • Solute Carrier Family 12, Member 2
  • Xenopus laevis


  • Slc12a2 protein, mouse
  • Sodium-Potassium-Chloride Symporters
  • Solute Carrier Family 12, Member 2
  • Prkwnk4 protein, mouse
  • Stk39 protein, mouse
  • Protein-Serine-Threonine Kinases