Akt phosphorylates and regulates the orphan nuclear receptor Nur77

Abstract

The immediate early gene NUR77 (also called NGFI-B) is required for T cell antigen receptor-mediated cell death and is induced to very high levels in immature thymocytes and T cell hybridomas undergoing apoptosis. The Akt (PKB) kinase is a key player in transduction of anti-apoptotic and proliferative signals in T cells. Because Nur77 has a putative Akt phosphorylation site at Ser-350, and phosphorylation of this residue is critical for the transactivation activity of Nur77, we investigated whether Akt regulates Nur77. Coimmunoprecipitation experiments showed the detection of Nur77 in Akt immune complexes, suggesting that Nur77 and Akt physically interact. We further show that Akt specifically phosphorylates Ser-350 of the Nur77 protein within its DNA-binding domain in vitro and in vivo in 293 and NIH 3T3 cells. Because phosphorylation of Ser-350 of Nur77 is critical for its function as a transcription factor, we examined the effect of Akt on this function. By using luciferase assay experiments, we showed that phosphorylation of Nur77 by Akt decreased the transcriptional activity of Nur77 by 50 -85%. Thus, we show that Akt interacts with Nur77 and inactivates Nur77 by phosphorylation at Ser-350 in a phosphatidylinositol 3-kinase-dependent manner, connecting the phosphatidylinositol 3-kinase-dependent Akt pathway and a nuclear receptor pathway.

Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt

“…Previous studies revealed that Akt phosphorylated Ser351 of Nur77, leading to the inhibition of Nur77 DNA binding, transactivation and apoptotic activities (Masuyama et al, 2001;Pekarsky et al, 2001). Akt mediates the anti-apoptotic effects of growth factors through PI3-K.…”

“…We, therefore, studied whether Akt, which is known to phosphorylate Nur77 (Masuyama et al, 2001;Pekarsky et al, 2001), was involved. First, we examined the effect of 3-Cl-AHPC on Akt activation in H460 cells, in which 3-Cl-AHPC-induced Nur77 nuclear export was rapid, occurring within 1-6 h (Li et al, 2000;Cao et al, 2004;Lin et al, 2004).…”

“…Thus, Akt activation inhibited MEKK1-DA-induced Nur77 nuclear export. Human Nur77 is phosphorylated by Akt at Ser351 (Ser350 in rat Nur77) (Masuyama et al, 2001;Pekarsky et al, 2001). To study whether the effect of CA-Akt was due to its phosphorylation of Nur77, Ser351 of Nur77 was replaced with alanine.…”

“…Thus, phosphorylation may play a role in regulating Nur77 subcellular trafficking. In fact, Nur77 is heavily phosphorylated in various cell types and phosphorylation regulates Nur77 functions (Fahrner et al, 1990;Hazel et al, 1991;Davis et al, 1993;Hirata et al, 1993;Weih et al, 1996;Katagiri et al, 2000;Masuyama et al, 2001;Pekarsky et al, 2001;Kolluri et al, 2003). Nur77 is phosphorylated at its N-terminal A/B domain by Jun N-terminal kinase (JNK) (Kolluri et al, 2003) and at Ser350 (Ser351 in human Nur77) by Akt (Masuyama et al, 2001;Pekarsky et al, 2001).…”

“…Nur77 is phosphorylated at its N-terminal A/B domain by Jun N-terminal kinase (JNK) (Kolluri et al, 2003) and at Ser350 (Ser351 in human Nur77) by Akt (Masuyama et al, 2001;Pekarsky et al, 2001). Nur77 phosphorylation by JNK and Akt modulates its DNA-binding and transactivation functions (Masuyama et al, 2001;Pekarsky et al, 2001;Kolluri et al, 2003). The role of phosphorylation in Nur77 nuclear export is demonstrated by the finding that nerve growth factor (NGF)-induced NGFI-B nuclear export requires the phosphorylation of NGFI-B at Ser105 (Ser140 in human Nur77) by MAP kinase pathway (Katagiri et al, 2000).…”