title: 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, 121-127]. 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 32P 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 Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulin-dependent 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.
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D047428:Protein Kinase Inhibitors; D051381:Rats; D012701:Serotonin / Q000378:metabolism; D014365:Tryptophan Hydroxylase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D012701:Serotonin; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D014365:Tryptophan Hydroxylase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease