The RAS-ERK pathway is known to play a pivotal role in differentiation, proliferation and tumour progression. Here, we show that ERK downregulates Forkhead box O 3a (FOXO3a) by directly interacting with and phosphorylating FOXO3a at Ser 294, Ser 344 and Ser 425, which
NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript consequently promotes cell proliferation and tumorigenesis. The ERK-phosphorylated FOXO3a degrades via an MDM2-mediated ubiquitin-proteasome pathway. However, the nonphosphorylated FOXO3a mutant is resistant to the interaction and degradation by murine double minute 2 (MDM2), thereby resulting in a strong inhibition of cell proliferation and tumorigenicity. Taken together, our study elucidates a novel pathway in cell growth and tumorigenesis through negative regulation of FOXO3a by RAS-ERK and MDM2.The constitutive activation of certain signal transduction cascades leads to the development of tumours and the resistance of tumours to clinical therapy 1,2 . The RAS-ERK pathway triggers one of these cascades and governs many important functions, such as cell fate, differentiation, proliferation and survival in invertebrate and mammalian cells 3,4 . Human tumours frequently overexpress RAS or harbour activated RAS with a point mutation, which contributes substantially to tumour cell growth, invasion and angiogenesis 1, 2 , 5 -8. Cell plasma membrane receptor tyrosine kinases activate RAS GTPases, and GTP-bound RAS activates A-RAF, B-RAF and RAF-1 (ref. 10,17,18,21 , but the E3 ubiquitin ligase responsible for FOXO3a degradation has yet to be identified. MDM2, an E3 ubiquitin ligase plays an important role in the development of multiple human cancers through degrading tumour suppressor proteins, such as p53, RB and E-cadherin [22][23][24][25] . In addition, MDM2 has been shown to be regulated by the RAS-ERK signalling pathway 26 and blocking ERK activity with an MEK1 inhibitor, U0126, reduces MDM2 expression in breast cancer cells 27 .Here, we identify a novel pathway involving the downregulation of FOXO3a expression by RAS-ERK and MDM2, which leads to promotion of cell growth and tumorigenesis. We show that ERK interacts with and phosphorylates FOXO3a at Ser 294, Ser 344 and Ser 425; phosphorylation of FOXO3a at these residues increases FOXO3a-MDM2 interaction and enhances FOXO3a degradation via an MDM2-dependent ubiquitin-proteasome pathway. The non-phosphorylated FOXO3a-mimic mutant, compared to the phosphorylated FOXO3a-mimic mutant, exhibits more resistance to the interaction and degradation by MDM2, resulting in a strong inhibition of cell proliferation in vitro and tumorigenesis in vivo.
RESULTS
ERK suppresses FOXO3a stability and induces its nuclear exclusionThe RAS-ERK is an essential oncogenic signalling cascade that promotes tumour cell growth and development 8,28 . It was known that other oncogenic kinases, AKT and IKK, (Fig. 1b, c). Similarly, using Erk small interference RNA (siRNA) to knockdown ERK protein expression level in HeLa cells (Fig. 1d), or trea...
