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-5 cells.
In previous studies, we showed that pretreatment of rat FRTL-5 thyroid cells with TSH, or other agents that increased intracellular cAMP, markedly potentiated DNA synthesis in response to insulin-like growth factor-I (IGF-I). In addition, we found that TSH pretreatment caused an increase in tyrosine phosphorylation of intracellular proteins including an unidentified 125-kDa 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 tyrosine phosphorylation. The present studies were undertaken to determine how tyrosine kinase activation followed by an increase in tyrosine phosphorylation 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 protein-tyrosine phosphatase activities in the cell lysates by the in vitro assay. Chronic treatment with TSH or (Bu)2-cAMP stimulated tyrosine kinase activity in the particulate fraction and protein-tyrosine phosphatase activity in the soluble fraction, suggesting that tyrosine kinase plays more important roles for a cAMP-dependent increase in tyrosine phosphorylation of intracellular proteins. The increased tyrosine kinase activity was sensitive to genistein, a potent tyrosine kinase inhibitor. Genistein abolished both the cAMP-dependent increase in tyrosine phosphorylation of the 125-kDa protein and the enhanced DNA synthesis induced by IGF-I in a similar concentration-dependent manner. The only tyrosine-phosphorylated protein associated with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase in response to cAMP was 125 kDa. In addition, we found that PI 3-kinase activity bound to p85 subunit significantly increased after (Bu)2cAMP treatment. These results suggested that cAMP stimulates PI 3-kinase through tyrosine phosphorylation of the 125-kDa protein. We then measured DNA synthesis in cells pretreated for 24 h with TSH or (Bu)2cAMP in the absence or presence of LY294002, a PI 3-kinase inhibitor, followed by treatment with IGF-I for 24 h. Presence of LY294002 during TSH or (Bu)2cAMP pretreatment completely abolished cAMP-dependent potentiation of DNA synthesis induced by IGF-I. These results suggest that in FRTL-5 cells cAMP activates genistein-sensitive tyrosine 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.
Nedachi
T
T
Department of Animal Sciences, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Japan.
Akahori
M
M
Ariga
M
M
Sakamoto
H
H
Suzuki
N
N
Umesaki
K
K
Hakuno
F
F
Takahashi
S I
SI
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D014638:Vanadates / Q000494:pharmacology
D002867:Chromones; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009025:Morpholines; D010750:Phosphoproteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D014638:Vanadates; D003994:Bucladesine; D007334:Insulin-Like Growth Factor I; D004247:DNA; D019833:Genistein; D000242:Cyclic AMP; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases