FOXO Transcription Factors Are Negatively Regulated by p38 Map Kinases Downstream of FLT3 Receptor Signaling.

The phosphoinositide 3-kinase (PI3K)-AKT/PKB signal transduction cascade is an evolutionarily conserved pathway regulating the FOXO subclass of Forkhead transcription factors (FOXO1, FOXO3a and FOXO4) downstream of insulin, cytokine and growth factor receptors. When PI3K phospholipid kinase activity is low, FOXO proteins localize to the nucleus where they induce expression of genes that promote programmed cell death (FasL, Bim, TRAIL), inhibit cell cycle progression (p27KIP1, Rb related family member p130), facilitate DNA repair (GADD45) and protect against oxidative damage (MnSOD). Following receptor stimulation, increased levels of PtdIns(3,4,5)P3 are generated at the plasma membrane resulting in PDK1-mediated activation of the serine/threonine kinase AKT/PKB. Subsequent phosphorylation of FOXO proteins by AKT/PKB results in their active sequestration in the cytoplasm through binding to 14-3-3 proteins, thereby blocking their action as transcription factors. Recently, it has become evident that FOXO proteins can also be regulated through other signaling pathways. One example is phosphorylation and activation of FOXO4 by stress kinases JNK/SAPK in response to oxidative stress (

Here, we report that p38 mitogen activated protein kinases (MAPK) negatively regulate FOXO transcription factors downstream of FLT3 receptor tyrosine kinase signaling. Stimulation of human wild-type FLT3 receptor with FLT3 ligand, or expression of constitutively active mutants FLT3-ITD or FLT3-D835Y in Ba/F3 cells resulted in activation of p38 MAPK and phosphorylation of its known substrate ATF2 on threonine residue 71 (Thr71). Transient luciferase reporter assays showed that expression of a constitutively active MKK3 or MKK6, upstream activators of p38 MAPK, inhibited FOXO-induced gene transcription. The p38 MAPK inhibitors SB202190 and SB203580 as well as dominant-negative p38 MAPK blocked FLT3-mediated inhibition of FOXO transcription activation, suggesting p38 MAPK is both necessary and sufficient for FOXO regulation. In vitro kinase assays demonstrated that FOXO proteins are directly phosphorylated by p38 MAPK, although different p38 MAPK family members (p38α, p38β 2, p38γ and p38δ) displayed distinct FOXO protein substrate specificity. In conclusion, our data demonstrate that FOXO proteins are novel substrates of p38 MAP kinases and activation of p38 MAPK through FLT3 receptor signaling represents an important pathway to attenuate FOXO target gene regulation.