PROTEIN-TYROSINE PHOSPHORYLATION AND CALCIUM SIGNALING IN THYROID FRTL-5 CELLS

We examined the importance of tyrosine kinase(s) on the ATP-evoked Ca2 + entry and DNA synthesis of thyroid FRTL-5 cells. ATP rapidly and tra nsiently tyrosine phosphorylated a 72-kDa protein(s). This phosphoryla tion was abolished by pertussis toxin and by the tyrosine kinase inhib itor genistein, and was dependent on Ca2+ entry. Pretreatment of the c ells with genistein did not affect the release of sequestered Ca2+, bu t the capacitative Ca2+ or Ba2+ entry evoked by ATP or thapsigargin wa s attenuated. Pretreatment of the cells with orthovanadate enhanced th e increase in intracellular free Ca2+ ([Ca2+](i)), whereas the Ba2+ en try was not increased. Phorbol 12-myristate 13-acetate (PMA) phosphory lated the same protein(s) as did ATP. Genistein inhibited the ATP-evok ed phosphorylation of MAP kinase and attenuated both the ATP-and the P MA-evoked DNA synthesis. However, genistein did not inhibit the ATP-ev oked expression of c-fos. Furthermore, genistein enhanced the ATP-evok ed release of arachidonic acid. Thus, ATP activates a tyrosine kinase via a Ca2+-dependent mechanism. A genistein-sensitive mechanism partic ipates, in part, in the ATP-evoked activation of DNA synthesis. Genist ein inhibits only modestly capacitative Ca2+ entry in FRTL-5 cells. (C ) 1998 Wiley-Liss, Inc.