Rapid turnover of tryptophan hydroxylase is driven by proteasomes in RBL2H3 cells, a serotonin producing mast cell line

Previously we demonstrated that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by ATP-dependent proteolysis in serotonin producing m ast cells [Hasegawa et at. (1995) FEBS Lett, 368, 151-154], We searched for the major proteases involved in the rapid degradation of TPH in RBL2H3 cel ls. Among various protease inhibitors tested, proteasome inhibitors MG115, MG101, MG132, and lactacystin effectively inhibited the intracellular degra dation of TPH, Administration of the proteasome inhibitors to cultured cell s caused more than a 5-fold accumulation of TPH, Administration of the inhi bitors together with cycloheximide stabilized the amount of TPH with no app reciable increase or decrease, Although MG-series proteasome inhibitors cou ld inhibit calpain, the involvement of calpain was excluded since the cyste ine protease inhibitor E-64-d, which acts on calpain, had no effect, Extrac ts of RBL2H3 cells were shown to contain a protease that digests TPH in an ATP-dependent manner and is sensitive to proteasome inhibitors. The ubiquit ination of TPH could be visualized by Western blot analysis using both anti -TPH and anti-ubiquitin antibodies, Based on these results, we conclude tha t 26S proteasomes are mainly involved in the degradation of TPH. In the rep orted amino acid sequences of TPH from various sources including human, rab bit, rat, and mouse, a PEST sequence that is widely shared among short-live d proteins has been recognized. It was noted that the PEST sequence lies wi thin the most conserved portion of the enzyme, the pteridine binding site.