tailieunhanh - Báo cáo Y học: Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line

We previously demonstrated inmast 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) (Tokyo) 2000,127, 121–127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turn-over, using non-neural TPH. | Eur. J. liiochem. 269 4780-4788 2002 FEBS 2002 doi Proteasome-driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells a serotonin producing mast cell line Yoshiko Iida1 Keiko Sawabe1 Masayo Kojima1 Kazuya Oguro1 2 Nobuo Nakanishi3 and Hiroyuki Hasegawa1 2 1 Department of Bioscience and 2Biotechnology Research Center Teikyo University of Science and Technology Yamanashi Japan 3Departments of Biochemistry Meikai University School of Dentistry Sakado Saitama Japan 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. Bỉochem Toko 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 .