Regulation of phorbol ester-induced Ras/Raf/Erk signaling pathway in EL4 cells

Shujie Han

doi:10.7273/000005819

The mechanisms of cellular responses to the tumor promoter phorbol 12-myristate 13-acetate (PMA) were examined in a panel of EL4 lymphoma cells. We hypothesized that 1) RasGRP1 confers the PMA-sensitive phenotype in EL4 cells; 2) integration of the PI3K/Akt signaling pathway at Raf-1 and feedback regulation of Raf-1 by Erk contribute to the differences between PMA-sensitive, -resistant and intermediate phenotypes; and 3) Raf kinase inhibitor protein (RKIP) is involved in regulating Erk activation, migration and invasion in EL4 cells. First, we characterized PMA-induced Ras/Raf-1/Erk activation in representative EL4 cell lines. We manipulated RasGRP1 levels using RasGRP siRNA in WT2 cells, and then tested PMA-induced Ras/Raf-1/Erk activation, proliferation, and IL-2 production. Based on these findings, we conclude that RasGRP1 confers the major features of the PMA-sensitive phenotype in EL4 cells. The second study examined the regulation of Ras/Raf/Erk signaling pathway by the PI3K/Akt and Erk pathways. Based on experiments using pharmacologic inhibitors, we conclude that negative regulation of Raf-1 at Ser259 by PI3K/Akt and auto-inhibitory feedback of Raf-1 at Ser289/296/301 by Erk are responsible in part for the differences in PMA-induced Raf/Erk activation between EL4 phenotypes. The third study was conducted to determine the potential contribution of RKIP to the enhanced metastasis of V7 cells. We used RKIP siRNA to knock down RKIP expression in V7 cells, and then assessed PMA-induced Raf-1/Erk phosphorylation, migration and invasion assay. Based on the results, we conclude that RKIP likely plays a role in the enhanced metastasis of V7 cells. Together these results suggest that multiple factors contribute to the distinct PMA responses in EL4 cells. RasGRP1 confers PMA-sensitive responses in EL4 cells; negative regulation of the Ras/Raf/Erk signaling pathway by PI3K/Akt at Raf-1 Ser259 and auto-inhibition of Raf-1 by Erk at Ser289/296/301 contribute to the distinct characters of PMA-sensitive, -resistant and intermediate phenotypes; and RKIP is, at least partially, responsible for enhanced migration and invasion of PMA-resistant cells. These studies illustrated how differences in the expression of key signaling proteins can either enhance or suppress events involved in tumor progression.

Regulation of phorbol ester-induced Ras/Raf/Erk signaling pathway in EL4 cells

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