Proteolytic cleavage of SPAK by a kidney protease (893.36)

Abstract

The Ste20 kinase SPAK is a major regulator of sodium reabsorption in the distal nephron. Recently, SPAK fragments have been detected in the mouse kidney, and particularly in the thick ascending limb of Henle (TAL). These truncated fragments have been demonstrated to inhibit the activity of the Na+‐K+‐2Cl‐ cotransporter, NKCC2, and thereby decrease sodium transport across the TAL. Here we show that mouse kidney lysates possess protease activity that leads to the specific cleavage of a SPAK, as we observed a precise and reproducible cleavage pattern with a GST‐SPAK fusion protein, but not a GST‐OSR1 fusion protein. This activity is not observed with brain, liver, or spleen lysates. The activity is insensitive to PMSF, leupeptin, aprotinin, and pepstatin, but inhibited by DTT and EDTA. In an effort to identify the protease, we performed ion exchange chromatography. The proteolytic activity was detected in an elution fraction corresponding to 250 mM NaCl. The fraction was subjected to MS/MS analysis and 1119 proteins were identified at 95% threshold. Among these proteins, we identified 44 peptidases and proteases. Gel permeation chromatography experiments determined that the protease is larger than 100 kDa. Effort will be made to determine the identity of the protein involved in the cleavage of SPAK and to identify the sites of cleavage.Grant Funding Source: RO1 DK0935010