Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3 ',5 '-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5cells

In previous studies, we showed that pretreatment of rat FRTL-6 thyroid cell s with TSH, or other agents that increased intracellular cAMP, markedly pot entiated DNA synthesis in response to insulinlike growth factor-I (IGF-I). In addition, we found that TSH pretreat ment caused an increase in tyrosine phosphorylation of intracellular proteins including an unidentified 125-kD a protein that was well correlated with the TSH-potentiating effect on DNA synthesis induced by IGF-I. These results suggested that cAMP amplified IGF I-dependent signals for cell growth through changes of cAMP-dependent tyros ine phosphorylation. The present studies were undertaken to determine how t yrosine kinase activation followed by an increase in tyrosine phosphorylati on is required for cAMP-dependent potentiation of DNA synthesis induced by IGF-I in this cell line. First of all, we measured tyrosine kinase or prote in-tyrosine phosphatase activities in the cell lysates by the in vitro assa y. Chronic treatment with TSH or (Bu)(2)-cAMP stimulated tyrosine kinase ac tivity in the particulate fraction and protein-tyrosine phosphatase activit y in the soluble fraction, suggesting that tyrosine kinase plays more impor tant roles for a cAMP-dependent increase in tyrosine phosphorylation of int racellular proteins. The increased tyrosine kinase activity was sensitive t o genistein, a potent tyrosine kinase inhibitor. Genistein abolished both t he cAMP-dependent increase in tyrosine phosphorylation of the 125-kDa prote in and the enhanced DNA synthesis induced by TGF-I in a similar concentrati on-dependent manner. The only tyrosine-phosphorylated protein associated wi th the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase in resp onse to cAMP was 125 kDa. In addition, we found that PI 3-kinase activity b ound to p85 subunit significantly increased after (Bu),cAMP treatment; Thes e results suggested that cAMP stimulates PI 3-kinase through tyrosine phosp horylation of the 125-kDa protein. We then measured DNA synthesis in cells pretreated for 24 h with TSH or (Bu)(2)cAMP in the absence or presence of L Y294002, a PI 3-kinase inhibitor, followed by treatment with IGF-I for 24 h . Presence of LY294002 during TSH or (Bu)2cAMP pretreatment completely abol ished cAMP-dependent potentiation of DNA synthesis induced by IGF-I. These results suggest that in FRTL-5 cells cAMP activates genistein-sensitive tyr osine kinases that in turn activate PI 3-kinase activity. These mechanisms appear to be necessary for cAMP-dependent potentiation of the DNA synthesis induced by IGF-I.