Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Am J Physiol Cell Physiol
2010 Sep 01;2993:C614-20. doi: 10.1152/ajpcell.00074.2010.
Show Gene links
Show Anatomy links
On the substrate recognition and negative regulation of SPAK, a kinase modulating Na+-K+-2Cl- cotransport activity.
Gagnon KB
,
Delpire E
.
???displayArticle.abstract???
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.
Alvarez-Leefmans,
Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes.
1988, Pubmed
Alvarez-Leefmans,
Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes.
1988,
Pubmed
Castrop,
Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance.
2008,
Pubmed
Ching,
Identification of an autoinhibitory domain of p21-activated protein kinase 5.
2003,
Pubmed
Creasy,
The Ste20-like protein kinase, Mst1, dimerizes and contains an inhibitory domain.
1996,
Pubmed
Crouch,
Immunohistochemical localization of the Na-K-Cl co-transporter (NKCC1) in the gerbil inner ear.
1997,
Pubmed
Darman,
A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1.
2002,
Pubmed
Delpire,
The mammalian family of sterile 20p-like protein kinases.
2009,
Pubmed
Delpire,
Genome-wide analysis of SPAK/OSR1 binding motifs.
2007,
Pubmed
Delpire,
Molecular cloning and chromosome localization of a putative basolateral Na(+)-K(+)-2Cl- cotransporter from mouse inner medullary collecting duct (mIMCD-3) cells.
1994,
Pubmed
Delpire,
Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter.
1999,
Pubmed
Edgcomb,
Variability in the pKa of histidine side-chains correlates with burial within proteins.
2002,
Pubmed
Falin,
Identification of regulatory phosphorylation sites in a cell volume- and Ste20 kinase-dependent ClC anion channel.
2009,
Pubmed
Gagnon,
A single binding motif is required for SPAK activation of the Na-K-2Cl cotransporter.
2007,
Pubmed
,
Xenbase
Gagnon,
Volume sensitivity of cation-Cl- cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4.
2006,
Pubmed
,
Xenbase
Gagnon,
Characterization of SPAK and OSR1, regulatory kinases of the Na-K-2Cl cotransporter.
2006,
Pubmed
,
Xenbase
Giménez,
Regulatory phosphorylation sites in the NH2 terminus of the renal Na-K-Cl cotransporter (NKCC2).
2005,
Pubmed
,
Xenbase
Glantschnig,
Mapping of MST1 kinase sites of phosphorylation. Activation and autophosphorylation.
2002,
Pubmed
Greger,
Ion transport mechanisms in thick ascending limb of Henle's loop of mammalian nephron.
1985,
Pubmed
Kennelly,
Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases.
1991,
Pubmed
Lee,
Crystal structure of domain-swapped STE20 OSR1 kinase domain.
2009,
Pubmed
Li,
SPAK kinase is a substrate and target of PKCtheta in T-cell receptor-induced AP-1 activation pathway.
2004,
Pubmed
Liedtke,
Regulation of chloride transport in epithelia.
1989,
Pubmed
Moriguchi,
WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1.
2005,
Pubmed
Mukherji,
Mutational analysis of secondary structure in the autoinhibitory and autophosphorylation domains of calmodulin kinase II.
1994,
Pubmed
Nolen,
Regulation of protein kinases; controlling activity through activation segment conformation.
2004,
Pubmed
Pang,
Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits.
2007,
Pubmed
Piechotta,
Cation chloride cotransporters interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1).
2002,
Pubmed
Piechotta,
Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl- cotransporter in the nervous system: evidence for a scaffolding role of the kinase.
2003,
Pubmed
,
Xenbase
Smith,
PKCdelta acts upstream of SPAK in the activation of NKCC1 by hyperosmotic stress in human airway epithelial cells.
2008,
Pubmed
Sung,
Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice.
2000,
Pubmed
Villa,
Structural insights into the recognition of substrates and activators by the OSR1 kinase.
2007,
Pubmed
Vitari,
The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases.
2005,
Pubmed
Zhao,
A conserved negative regulatory region in alphaPAK: inhibition of PAK kinases reveals their morphological roles downstream of Cdc42 and Rac1.
1998,
Pubmed
Zhou,
Crystal structure of the TAO2 kinase domain: activation and specificity of a Ste20p MAP3K.
2004,
Pubmed
Zhu,
A single pair of acidic residues in the kinase major groove mediates strong substrate preference for P-2 or P-5 arginine in the AGC, CAMK, and STE kinase families.
2005,
Pubmed
