Short forms of SPAK in the kidney are created by Dnpep-mediated proteolytic cleavage [Cell Biology]

August 27th, 2014 by Markadieu, N., Rios, K., Spiller, B. W., McDonald, W. H., Welling, P. A., Delpire, E.

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 towards SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase (Dnpep). Presence of the protease was verified in the active fractions and recombinant Dnpep 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 towards the Na+-K+-2Cl- cotransporter, NKCC2.