title: Short forms of Ste20-related proline/alanine-rich kinase (SPAK) in the kidney are created by aspartyl aminopeptidase (Dnpep)-mediated proteolytic cleavage.
abstract: 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.
authors.affiliation: From the Departments of Anesthesiology.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Markadieu
authors.affiliation: From the Departments of Anesthesiology.
authors.forename: Kerri
authors.initials: K
authors.lastname: Rios
authors.affiliation: Pathology, and.
authors.forename: Benjamin W
authors.initials: BW
authors.lastname: Spiller
authors.affiliation: Biochemistry and the Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and.
authors.forename: W Hayes
authors.initials: WH
authors.lastname: McDonald
authors.affiliation: the Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Welling
authors.affiliation: From the Departments of Anesthesiology, eric.delpire@vanderbilt.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Delpire
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001794:Blood Pressure; D003001:Cloning, Molecular; D043384:Glutamyl Aminopeptidase / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D007679:Kidney Medulla / Q000378:metabolism; D013058:Mass Spectrometry; D045726:Metalloproteases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D012964:Sodium / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D011993:Recombinant Fusion Proteins; D012964:Sodium; C486407:Stk39 protein, mouse; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human; D045726:Metalloproteases; D043384:Glutamyl Aminopeptidase
keywords: Metalloprotease; Protein Kinase; Renal Physiology; Signal Transduction; Sodium Transport