Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1
| Autor: | Alberto C. Vitari, Nick A. Morrice, Jacob Thastrup, Håkan K. R. Karlsson, Maria Deak, Fatema H. Rafiqi, Dario R. Alessi |
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| Rok vydání: | 2006 |
| Předmět: |
Threonine
Sodium-Potassium-Chloride Symporters Peptide Biology Protein Serine-Threonine Kinases Kidney Biochemistry Substrate Specificity Minor Histocompatibility Antigens WNK Lysine-Deficient Protein Kinase 1 Serine Humans Solute Carrier Family 12 Member 2 Phosphorylation Protein kinase A Molecular Biology Cells Cultured Alanine chemistry.chemical_classification Binding Sites Sequence Homology Amino Acid Kinase Activator (genetics) Osmolar Concentration Intracellular Signaling Peptides and Proteins Cell Biology WNK1 WNK4 Enzyme Activation chemistry Hypertension Mutation Hyperkalemia Research Article |
| Zdroj: | The Biochemical journal. 397(1) |
| ISSN: | 1470-8728 |
| Popis: | The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na + –K + –2Cl − co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon9s hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr 175 /Thr 179 /Thr 184 in shark or Thr 203 /Thr 207 /Thr 212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na + –Cl − co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon9s syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4). |
| Databáze: | OpenAIRE |
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