We have reported that pretreatment of rat FRTL-5 thyroid cells with thyrotr
opin (TSH) markedly potentiates the mitogenic response to insulin-like grow
th factor-I (IGF-I). The present study was undertaken to determine whether
the augmentation by cAMP of IGF-I-dependent tyrosine phosphorylation of kno
wn IGF-I receptor substrates plays an important role in the cAMP-dependent
potentiation of DNA synthesis induced by IGF-I. Pretreatment with TSH or di
butyryl cAMP did not affect the IGF-I-dependent tyrosine phosphorylation of
insulin receptor substrate-1 (IRS-1), In contrast, cAMP pretreatment poten
tiated the tyrosine phosphorylation of IRS-2 induced by IGF-I, but did not
affect the amount of IRS-2, We found that the IGF-I-dependent tyrosine phos
phorylation of 66 kDa Shc (Src homology collagen) was markedly increased by
cAMP pretreatment, and that this change was mainly due to an increase in t
he levels of 66 kDa Shc protein. Under these conditions, cAMP pretreatment
significantly increased binding of Grb2 (growth-factor-receptor-bound prote
in 2) to Shc in response to IGF-I, and activation of MAP kinase (mitogen-ac
tivated protein kinase) induced by IGF-I was also enhanced by cAMP. The pre
sence of PD98059, an inhibitor of MEK (MAP-kinase/Erk kinase), during treat
ment with IGF-I partially inhibited the cAMP-dependent augmentation of DNA
synthesis in response to IGF-I. On the other hand, cAMP pretreatment increa
sed binding of the phosphoinositide 3-kinase (PI 3-kinase) p85 subunit to I
RS-2, which was reflected in PI 3-kinase activity. LY294002, a PI 3-kinase
inhibitor, strongly depressed IGF-I-dependent DNA synthesis after pretreatm
ent with and without TSH or dibutyryl cAMP. Our results suggest that the in
teraction between cAMP-dependent and IGF-I-dependent pathways leads to an a
ugmentation of cell proliferation, which is mediated, at least in part, thr
ough the MAP kinase and PI 3-kinase signalling pathways. These effects are
mediated by changes in tyrosine phosphorylation of IGF-I receptor substrate
s, including IRS-2 and Shc.