Submitted on April 4, 2008
Revised on August 24, 2008
Accepted on August 25, 2008
An evolutionarily conserved N-terminal Sgk1 variant with enhanced stability and improved function
Nandita S Raikwar1, Peter M. Snyder2, and Christie P Thomas2*
1 UNiversity of Iowa
2 University of Iowa
* To whom correspondence should be addressed. E-mail: christie-thomas{at}uiowa.edu.
Sgk1 is an aldosterone-induced kinase that regulates epithelial sodium channel (ENaC)-mediated Na+ transport in the collecting duct and connecting tubule of the kidney. The NH2-terminus of Sgk1 contains instability motifs that direct the ubiquitination of sgk1 resulting in a rapidly degraded protein. By bioinformatic analysis we identified a 5' variant alternate transcript of human Sgk1 (Sgk1_v2) that is widely expressed, is conserved from rodent to man and is predicted to encode an Sgk1 isoform, Sgk1_i2, with a different NH2-terminus. When expressed in HEK293 cells, Sgk1_i2 was more abundant than Sgk1 because of an increased protein half-life and this correlated with reduced ubiquitination of Sgk1_i2 and enhanced surface expression of ENaC. Immunocytochemical studies demonstrated that in contrast to Sgk1, Sgk1_i2 is preferentially targeted to the plasma membrane. When co- expressed with ENaC subunits in FRT epithelia, Sgk1_i2 had a significantly greater effect on amiloride-sensitive Na+ transport as compared to Sgk1. Together the data demonstrate that a conserved N-terminal variant of Sgk1 shows improved stability, enhanced membrane association and greater stimulation of epithelial Na+ transport in a heterologous expression system.
