Phorbol Esters Stimulate Ubiquitination of NCC By Activating ERK1/2

Abstract

The sodium chloride co‐transporter (NCC) is the principal salt‐absorptive pathway in the distal convoluted tubule (DCT). We have described a pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl‐releasing protein 1 (RasGRP1), activating ERK1/2 MAPK and decreasing NCC activity. We have also shown that PEs trigger the ubiquitination and internalization of NCC. To test whether these findings are related, we examined the ability of the PE TPA to increase NCC ubiquitination while inhibiting the RasGRP1/ERK1/2 pathway. mDCT cells were treated with 100nM TPA and the MEK1/2 inhibitor U0126 for 15 minutes. Lysis, immunoprecipitation for NCC, and immunoblotting for ubiquitin showed that U0126 prevented any PE‐induced increase in ubiquitination (no change compared to a 493±4% increase in ubiquitination with TPA alone). To examine RasGRP1's role, its expression was silenced using siRNA. These cells showed no increase in ubiquitination compared to RasGRP1 knockdown alone. These data indicate that TPA's ability to enhance NCC ubiquitination is dependent upon activation of RasGRP1 and ERK1/2.