Wh. Zheng et al., Insulin-like growth factor-1-induced phosphorylation of the Forkhead family transcription factor FKHRL1 is mediated by Akt kinase in PC12 cells, J BIOL CHEM, 275(50), 2000, pp. 39152-39158
The Forkhead family transcription factor FKHRL1, a mammalian homolog of DAF
16 in the nematode Caenorhabditis elegans, is an inducer of apoptosis in it
s unphosphorylated form and was recently reported as a substrate of Akt kin
ases. Insulin-like growth factor (IGF-I) is a potent stimulant of Akt kinas
e, leading to inhibition of the apoptotic pathway. In this study, we charac
terized the phosphorylation of FKHRL1 induced by IGF-1 in PC12 cells and va
rious neuronal cell types and examined the potential role of Akt in this re
gard. IGF-I rapidly induced the phosphorylation of Akt and FKHRL1 in PC12 c
ells. The phosphorylation of Akt and FRHRL1 induced by 10 nM IGF-1 was inhi
bited by the phosphatidylinositide S-kinase (PI3K) inhibitors wort-mannin (
0.25-2 muM) and LY294002 (12.5-100 muM), but not by the MEK inhibitor PD980
59 (50 muM) or the p70 S6 kinase pathway inhibitor rapamycin (50 nM), sugge
sting that the phosphorylation of FKHRL1 induced by IGF-I is mediated by th
e PI3K pathway. As observed for IGF-1, an in vitro kinase assay with purifi
ed active Akt kinase demonstrated that the kinase is capable of directly ph
osphorylating FKHRL1 at Thr(32) and Ser(253), leading to inhibition of its
pro-apoptotic properties. Moreover, transient expression of constitutively
active Akt (MS-Akt, where MS is a myristylation signal) increased the phosp
horylation of FKHRL1, whereas the expression of kinase-dead Akt (M179A Akt)
attenuated the phosphorylation of FKHRL1 induced by 10 nM IGF-1 in PC12 ce
lls. Interestingly, FKHRL1 co-immunoprecipitated with Akt in PC12 cells, in
dicating that these two proteins can associate in these cells. As IGF-1 als
o induced the phosphorylation of FKHRL1 in primary cortical and cerebellar
neuronal cultures, these data, taken together, demonstrate that IGF-1, acti
ng via the PI3K/Akt kinase pathway, can regulate the phosphorylation of FKH
RL1, leading to inhibition of this apoptotic transcription factor in neuron
al cells.