Identification of Protein Kinase C and Its Multiple Isoforms in FRTL-5 ThyroidCells

摘要

Protein kinase C (PKC) has been implicated as an important regulator of signaltransduction in the FRTL-S thyroid cell line hut little is known about its isoforms in this cell line. In the present investigation we characterized the activation of PKC by measuring the enzyme activity and identifying its isoforms in both cytosol and membrane fractions. Phorbol 12-myristate 13-acetate (PMA) was used as a PKC activator in this study. PKC activity assay revealed that PMA (300 nM) induced a rapid translocation from cytosol to membrane within 1 min and led to an almost complete translocation within 15 min. Multiple PKC isoforms were examined by Western blot analysis with specific antibodies against alpha, beta, gamma, delta, epsilon, and zeta isoforms. PKC alpha, beta, epsilon, and zeta were identified in this cell line, but PKC beta and gamma were not. Exposure of the cells to PMA (300 nM) for 5 to 30 min led to the translocation of PKC alpha, beta, and epsilon from the cytosol to the membrane fraction, while PKC zeta was not affected. Treatment with PMA (300 nM) for 24 h resulted in the down-regulation of PKC alpha, beta, and epsilon but not PKC zeta. This study demonstrates for the first time direct evidence for the activation of PKC, and expression and distribution of its isoforms in FRTL-5 thyroid cells.