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 mast cells [Hasegawa et al. (1995) FEBS Lett. 368, 151-154]. We searched for the major proteases involved in the rapid degradation of TPH in RBL2H3 cells. Among various protease inhibitors tested, proteasome inhibitors MG115, MG101, MG132, and lactacystin effectively inhibited the intracellular degradation of TPH. Administration of the proteasome inhibitors to cultured cells caused more than a 5-fold accumulation of TPH. Administration of the inhibitors together with cycloheximide stabilized the amount of TPH with no appreciable increase or decrease. Although MG-series proteasome inhibitors could inhibit calpain, the involvement of calpain was excluded since the cysteine protease inhibitor E-64-d, which acts on calpain, had no effect. Extracts of RBL2H3 cells were shown to contain a protease that digests TPH in an ATP-dependent manner and is sensitive to proteasome inhibitors. The ubiquitination of TPH could be visualized by Western blot analysis using both anti-TPH and anti-ubiquitin antibodies. Based on these results, we conclude that 26S proteasomes are mainly involved in the degradation of TPH. In the reported amino acid sequences of TPH from various sources including human, rabbit, rat, and mouse, a PEST sequence that is widely shared among short-lived proteins has been recognized. It was noted that the PEST sequence lies within the most conserved portion of the enzyme, the pteridine binding site.