Proteasome‐driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line

Abstract

We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S-proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127,[121][122][123][124][125][126][127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non-neural TPH. The proteasome-driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32 P into a 53-kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti-TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell-free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P-815 cells was inhibited by protein kinase inhibitors KN-62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca 2+ /calmodulindependent protein kinase II in the presence of Ca 2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca 2+ /calmodulindependent protein kinase II was digested more rapidly in the cell-free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome-driven TPH degradation.Keywords: tetrahydrobiopterin; CaM kinase II; proteasome target; ubiquitin ligase; enzyme turnover.Tryptophan hydroxylase (TPH, EC 1.14.16.4), a member of a family of pterin-dependent aromatic amino acid hydroxylases [1], catalyzes the conversion of L-tryptophan to 5-hydroxy-L-tryptophan. This reaction is the initial and rate-limiting step in the biosynthesis of serotonin [2][3][4][5]. TPH has been extensively purified from various sources such as bovine pineal gland [6], mouse mastocytoma [7,8], and mammalian brains [9][10][11]. Physicochemical, enzymic and immunochemical properties differed between TPHs of neural and non-neural tissue origin, and it is accepted that neural TPH might be a different entity from the non-neural enzyme [8,10,12,13]. Complimentary DNAs of TPH have been cloned from various sources but no differences or only trivial variation in amino acid sequences were found among them [14][15][16][17][18][19]. The molecular basis of differences between the neural and non-neural enzymes has not yet been explained.Both types of cytosolic environment should be studied further to detect differences in the control of gene expression, post-translational modification, and turnover of the enzyme protein in a tissue-specific way.We have demonstrated with RBL2H3, an established cell line that expresses TPH in culture while retaining many of the characteristics of mast cells, that: (a) cellular TPH activity was ...

The cutaneous serotoninergic/melatoninergic system: securing a place under the sun

“…These findings indicate extensive turnover of the enzyme in skin, as described in other models (102). It is thought that high molecular weight TPH-like species may represent ubiquitinated forms of TPH whereas low molecular weight TPH-like species may represent degradation products (102). TPH enzymatic activity has been detected in extracts of cutaneous melanoma cells (23) and in skin melanocytes, keratinocytes, and fibroblasts (Slominski et al, unpublished results), while metabolic products hydroxytryptophan and serotonin were identified in melanoma cells by liquid chromatography-mass spectrometry (LS/MS).…”

“…TPH immunoreactivities detected include forms with the size expected for the intact enzyme, as well as higher or lower molecular weight forms. These findings indicate extensive turnover of the enzyme in skin, as described in other models (102). It is thought that high molecular weight TPH-like species may represent ubiquitinated forms of TPH whereas low molecular weight TPH-like species may represent degradation products (102).…”

Posttranscriptional regulation of adrenal TH gene expression contributes to the maladaptive responses triggered by insulin-induced recurrent hypoglycemia

“…However, some reports indicate that the phosphorylation of hydroxylases (including TH) is a prerequisite for proteasome‐driven protein degradation in vitro (Iida et al. ; Kawahata et al. ; Nakashima et al.…”

Late Developmental Stage-Specific Role of Tryptophan Hydroxylase 1 in Brain Serotonin Levels

“…Two days after transfection, cells were harvested, and the amount of TPH1 or TPH2 protein fused to the histidine hexamer was evaluated by Western blot analysis using antihistidine-hexamer antibody (Sigma, St. Louis, MO). Then, the TPH activity was determined through a three-step procedure (Iida et al, 2002).…”