Proteasomeâdriven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line
2002
Iida, Yoshiko | Sawabe, Keiko | Kojima, Masayo | Oguro, Kazuya | Nakanishi, Nobuo | Hasegawa, Hiroyuki
We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26Sâproteasomes [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 nonâneural TPH. The proteasomeâdriven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32P into a 53âkDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using antiâTPH 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 cellâfree proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma Pâ815 cells was inhibited by protein kinase inhibitors KNâ62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca2+/calmodulinâdependent protein kinaseâII in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulinâdependent protein kinaseâII was digested more rapidly in the cellâfree proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasomeâdriven TPH degradation.
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