Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

Nicolas Markadieu,Kerri Rios,Benjamin W Spiller,W Hayes Mcdonald,Paul A Welling,Eric Delpire

doi:10.1074/jbc.m114.604009

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.

Highlights

C-terminal SPAK fragments are found in kidney medulla
In the kidney medulla several smaller SPAK fragments are observed by Western blot analysis, and these fragments have been postulated to act as negative regulators of OSR1 function in the thick ascending limb of Henle [14, 16, 18]
A GST fusion protein comprising of the entire open reading frame of SPAK flanked by 5Ј FLAG and 3Ј HA epitopes (Fig. 1B) was incubated with a kidney lysate, and the SPAK reactants were subjected to Western blot analysis with either FLAG or HA antibodies or an antibody that recognizes the C-terminal domain of SPAK

Summary

Background

C-terminal SPAK fragments are found in kidney medulla. Results: We identified Dnpep as the protease responsible for SPAK cleavage, identified the sites of cleavage, and showed unusual preference for ␣ helices. In the kidney medulla several smaller SPAK fragments are observed by Western blot analysis, and these fragments have been postulated to act as negative regulators of OSR1 function in the thick ascending limb of Henle [14, 16, 18]. If these fragments were to inhibit OSR1 function, their absence in the knock-out would lead to increased OSR1 and hyperphosphorylation of NKCC2. We considered alternative mechanisms for the production of the fragments and considered the possibility that SPAK is modified by proteolytic cleavage in the kidney

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION