MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling
Wei Wang, … , David D. Moore, Feng Yang
Wei Wang, … , David D. Moore, Feng Yang
Published January 28, 2019
Citation Information: J Clin Invest. 2019;129(3):1015-1029. https://doi.org/10.1172/JCI97712.
View: Text | PDF
Research Article Cell biology Oncology Article has an altmetric score of 75

MAPK4 overexpression promotes tumor progression via noncanonical activation of AKT/mTOR signaling

Abstract

MAPK4 is an atypical MAPK. Currently, little is known about its physiological function and involvement in diseases, including cancer. A comprehensive analysis of 8887 gene expression profiles in The Cancer Genome Atlas (TCGA) revealed that MAPK4 overexpression correlates with decreased overall survival, with particularly marked survival effects in patients with lung adenocarcinoma, bladder cancer, low-grade glioma, and thyroid carcinoma. Interestingly, human tumor MAPK4 overexpression also correlated with phosphorylation of AKT, 4E-BP1, and p70S6K, independent of the loss of PTEN or mutation of PIK3CA. This led us to examine whether MAPK4 activates the key metabolic, prosurvival, and proliferative kinase AKT and mTORC1 signaling, independent of the canonical PI3K pathway. We found that MAPK4 activated AKT via a novel, concerted mechanism independent of PI3K. Mechanistically, MAPK4 directly bound and activated AKT by phosphorylation of the activation loop at threonine 308. It also activated mTORC2 to phosphorylate AKT at serine 473 for full activation. MAPK4 overexpression induced oncogenic outcomes, including transforming prostate epithelial cells into anchorage-independent growth, and MAPK4 knockdown inhibited cancer cell proliferation, anchorage-independent growth, and xenograft growth. We concluded that MAPK4 can promote cancer by activating the AKT/mTOR signaling pathway and that targeting MAPK4 may provide a novel therapeutic approach for cancer.

Authors

Wei Wang, Tao Shen, Bingning Dong, Chad J. Creighton, Yanling Meng, Wolong Zhou, Qing Shi, Hao Zhou, Yinjie Zhang, David D. Moore, Feng Yang

×

Figure 4

MAPK4 interacts with AKT and is a novel T308 kinase.

Options: View larger image (or click on image) Download as PowerPoint
MAPK4 interacts with AKT and is a novel T308 kinase. (A) FLAG/His-tagged...
(A) FLAG/His-tagged MAPK4 and the MAPK42A mutant were overexpressed in PNT1A cells and purified using Ni-NTA column followed by immunoprecipitation using anti-FLAG M2 affinity gel. In vitro kinase assay was performed using a commercially available recombinant AKT1. AKT phosphorylation was detected by Western blots. MAPK42A carries KVAVAA in place of the KVAVKK ATP binding motif. Coomassie blue staining reveals that the purified FLAG/His-tagged MAPK4 (with a calculated molecular weight of 69.6 kDa) contains one major band around 70 kDa. F/H: 2× FLAG and 10× His tag. (B) MAPK4 and the MAPK42A and MAPK45A mutants were overexpressed in HCT116 PDK1-KO cells. AKT phosphorylation was detected by Western blots. MAPK45A carries AAAAAA in place of the KVAVKK ATP binding motif. Coimmunoprecipitation (co-IP) assays reveal (C) ectopically overexpressed AKT1 binding to ectopically overexpressed MAPK4 in HEK293T cells, and (D) endogenous AKT1 binding to endogenous MAPK4 in VCaP cells. (E) Pull-down assay on the binding between purified GST-MAPK4 protein and purified AKT1 as well as endogenous AKT in the HEK293T, H1299, and HepG2 cell lysates. GST was used as control. GST-MAPK4 and GST were overexpressed in E. coli and purified using the glutathione sepharose beads. Coomassie blue staining confirms major bands of around 90 kDa and 26 kDa in the purified GST-MAPK4 and GST, respectively. (F) BiFC assay for AKT association with MAPK4 in cytoplasm. Hela cells were cotransfected with YN-MAPK4 and YC-AKT1 or YC control vectors. Forty-eight hours later, the cells were fixed, counterstained with DAPI, and imaged for YFP fluorescence to indicate MAPK4-AKT1 interaction. Original magnification: ×400. (G) Three different MK5 siRNAs were transfected into control and MAPK4-overexpressing HEK293T cells. Western blots were used to confirm MK5 knockdown and compare AKT phosphorylation. Ctrl: control. Data are representative of at least 3 independent experiments.