Down-regulation of 14-3-3η protein by RNAi increases stability of exogenous tyrosine hydroxylase in PC12D cells

Abstract

The key portion of the N-terminus of human tyrosine hydroxylase type 1 (hTH1) that affects the stability of the molecule in mammalian cells has not been clarified except for the observation that the sequence up to Ala⁵² is involved. Therefore, this study was performed to map more precisely the N-terminal sequence that predominantly contributes to the stability of hTH1. The results obtained by using N-terminus-deleted hTH1 mutants identified the sequence up to Ala²³ as exerting the effect. Next, in order to clarify the effect, PC12D cells exogenously expressing wild-type hTH1 or the mutants were established. Then, 14-3-3η proteins in the cells were down-regulated by using the RNAi method, because 14-3-3η can bind Ser¹⁹-phosphorylated hTH1. The down-regulation of 14-3-3η proteins in PC12D cells exogenously expressing hTH1 enhanced the stability of the wild-type enzyme and that of the mutant lacking the N-terminus up to Ala²³. However, the stability of the mutant was reduced compared to that of the wild-type enzyme. The stability of the mutant with the N-terminus deleted up to Glu⁴³ was not affected by the down-regulation of 14-3-3η. These results suggest that the 14-3-3η protein regulates hTH1 stability by acting on the N-terminus up to Ala²³. [J Physiol Sci. 2008;58 Suppl:S153]