Short forms of Ste20-related proline/alanine-rich kinase (SPAK) in the kidney are created by aspartyl aminopeptidase (Dnpep)-mediated proteolytic cleavage.

Markadieu N, Rios K, Spiller BW, McDonald WH, Welling PA, Delpire E
J Biol Chem. 2014 289 (42): 29273-84

PMID: 25164821 · PMCID: PMC4200278 · DOI:10.1074/jbc.M114.604009

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

MeSH Terms (21)

Amino Acid Sequence Animals Blood Pressure Cloning, Molecular Glutamyl Aminopeptidase Humans Kidney Kidney Medulla Mass Spectrometry Metalloproteases Mice Mice, Inbred C57BL Molecular Sequence Data Oocytes Protein-Serine-Threonine Kinases Protein Binding Protein Structure, Secondary Recombinant Fusion Proteins Signal Transduction Sodium Xenopus laevis

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