👤 Elizabeth V Wattenberg

Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways. Show less

We have capitalized on the unique properties of the skin tumor promoter palytoxin, which does not activate protein kinase C, to investigate alternative mechanisms by which major signaling molecules can be modulated during carcinogenesis. We report here that palytoxin activates extracellular signal-regulated kinase (ERK) through a novel mechanism that involves inactivation of an ERK phosphatase in keratinocytes derived from initiated mouse skin (308 cells). Use of U0126 revealed that palytoxin requires the ERK kinase MEK to stimulate ERK activity, although palytoxin did not activate MEK. We found that 308 keratinocytes highly express mitogen-activated protein kinase phosphatase-3 (MKP-3), which selectively inactivates ERK. Palytoxin induced the loss of MKP-3 in a manner that corresponded to increased ERK phosphorylation. Complementary studies showed that sustained expression of exogenous MKP-3 inhibited palytoxin-stimulated ERK activation. As is characteristic of initiated keratinocytes, 308 cells express activated H-Ras. To investigate whether expression of oncogenic Ras is key to palytoxin-stimulated ERK activation, we determined how palytoxin affected ERK and MKP-3 in MCF10A human breast epithelial cells and in H-ras MCF10A cells, which stably express activated H-Ras. Palytoxin did not affect ERK activity in MCF10A cells, which had no detectable MKP-3. Like 308 cells, H-ras MCF10A cells highly express MKP-3. Strikingly, palytoxin stimulated ERK activity and induced a corresponding loss of MKP-3 in H-ras MCF10A cells. These studies indicate that in initiated cells palytoxin unleashes ERK activity by down-regulating MKP-3, an ERK inhibitor, and further suggest that MKP-3 may be a vulnerable target in cells that express oncogenic Ras. Show less