An evolutionarily conserved N-terminal Sgkl variant with enhanced stability and improved function

摘要

Sgkl 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 NH_2 terminus of Sgkl contains instability motifs that direct the ubiquitination of Sgkl resulting in a rapidly degraded protein. By bioinformatic analysis, we identified a 5' variant alternate transcript of human Sgkl (Sgkl_v2) that is widely expressed, is conserved from rodent to humans, and is predicted to encode an Sgkl isoform, Sgkl_i2, with a different NH_2 terminus. When expressed in HEK293 cells, Sgkl_i2 was more abundant than Sgkl because of an increased protein half-life and this correlated with reduced ubiquitination of Sgkl_i2 and enhanced surface expression of ENaC. Immunocytochemical studies demonstrated that in contrast to Sgkl, Sgkl_i2 is preferentially targeted to the plasma membrane. When coexpressed with ENaC subunits in FRT epithelia, Sgkl_i2 had a significantly greater effect on amiloride-sensitive Na~+ transport compared with Sgkl. Together, the data demonstrate that a conserved NH_2-terminal variant of Sgkl shows improved stability, enhanced membrane association, and greater stimulation of epithelial Na~+ transport in a heterologous expression system.