Insulin-like Growth Factor-1-induced Phosphorylation of the Forkhead Family Transcription Factor FKHRL1 Is Mediated by Akt Kinase in PC12 Cells

Abstract

The Forkhead family transcription factor FKHRL1, a mammalian homolog of DAF16 in the nematode Caenorhabditis elegans, is an inducer of apoptosis in its unphosphorylated form and was recently reported as a substrate of Akt kinases. Insulin-like growth factor (IGF-1) is a potent stimulant of Akt kinase, leading to inhibition of the apoptotic pathway. In this study, we characterized the phosphorylation of FKHRL1 induced by IGF-1 in PC12 cells and various neuronal cell types and examined the potential role of Akt in this regard. IGF-1 rapidly induced the phosphorylation of Akt and FKHRL1 in PC12 cells. The phosphorylation of Akt and FKHRL1 induced by 10 nm IGF-1 was inhibited by the phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (0.25-2 microm) and LY294002 (12.5-100 microm), but not by the MEK inhibitor PD98059 (50 microm) or the p70 S6 kinase pathway inhibitor rapamycin (50 nm), suggesting that the phosphorylation of FKHRL1 induced by IGF-1 is mediated by the PI3K pathway. As observed for IGF-1, an in vitro kinase assay with purified active Akt kinase demonstrated that the kinase is capable of directly phosphorylating 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 phosphorylation of FKHRL1, whereas the expression of kinase-dead Akt (M179A Akt) attenuated the phosphorylation of FKHRL1 induced by 10 nm IGF-1 in PC12 cells. Interestingly, FKHRL1 co-immunoprecipitated with Akt in PC12 cells, indicating that these two proteins can associate in these cells. As IGF-1 also induced the phosphorylation of FKHRL1 in primary cortical and cerebellar neuronal cultures, these data, taken together, demonstrate that IGF-1, acting via the PI3K/Akt kinase pathway, can regulate the phosphorylation of FKHRL1, leading to inhibition of this apoptotic transcription factor in neuronal cells.

Downregulation of Bim by brain-derived neurotrophic factor activation of TrkB protects neuroblastoma cells from paclitaxel but not etoposide or cisplatin-induced cell death

“…38 It is predominantly regulated by FKHRL1, 21,[23][24][25][26] and the molecular mechanism associated with FKHRL1 regulation has largely been characterized in response to growth factor signaling via the evolutionarily conserved PI3K/Akt survival signaling pathway. [16][17][18][19]39 Using pharmacological inhibitors we found that FKHRL1 was regulated by the PI3K pathway. This is consistent with what has been reported for BDNF activation of endogenous TrkB receptors in SY5Y NB cells by Zhu et al 40 Moreover, a PI3K inhibitor did not block the BDNFinduced reduction of Bim.…”

“…10 Activated Akt phosphorylates and inhibits several proapoptotic proteins, such as Bad, caspase-9 and the Forkhead transcription factors that include FKHRL1, FKHR and AFX, leading to cell survival. [11][12][13][14][15] Although several growth factors (such as IGF-I, TGF, EPO, PDGF) [16][17][18][19] have been found to induce FKHRL1 phosphorylation via the Akt pathway, the role of Akt in BDNF regulation of FKHRL1 has not been explored. Forkhead transcription factor-induced cell death appears to be mediated by transcriptional regulation of a number of genes including Fas ligand (Fasl) 15,20 and Bim, a BH3 only domain protein that modulates the intrinsic mitochondrial death pathway.…”

Nerve Growth Factor Uses Ras/ERK and Phosphatidylinositol 3-Kinase Cascades to Up-regulate theN-Methyl-d-aspartate Receptor 1 Promoter

“…Another potential mediator may be the forkhead family of transcription factors. Studies of their roles in neuronal differentiation are just beginning, and recently it was shown that insulin-like growth factor I induces phosphorylation of FKHRL1 forkhead protein via PKB in PC12 cells (46). Nevertheless, how the PI3K signaling regulates the NR1 promoter still remains unsolved.…”

SHIP-2 and PTEN Are Expressed and Active in Vascular Smooth Muscle Cell Nuclei, but Only SHIP-2 Is Associated with Nuclear Speckles

“…The 5-phosphatase activity may switch PtdIns(3,4,5)P 3 -dependent signals to PtdIns(3,4)P 2 and consequently regulate nuclear PKB targets. In contrast, the 3-phosphatase activity should attenuate these signals and down-regulate nuclear targets of PKB, such as the forkhead family transcription factors, FKHR, FKHRL1 and AFX, that control the expression of pro-and anti-apoptotic genes (37,38).…”