Editors' ChoiceCell Biology

Connecting Energy Status to Proliferation

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Science Signaling  29 Oct 2013:
Vol. 6, Issue 299, pp. ec265
DOI: 10.1126/scisignal.2004848

Cells integrate various extracellular signals and information about cellular status to control proliferation. Signaling by the kinase cascade B-RAF to MEK to ERK stimulates cellular proliferation, and aberrant activation of this pathway can lead to cancer. In particular, an activating mutation in B-RAF is a common cause of melanoma, and therapies targeting this kinase are effective. However, some patients develop another form of skin cancer called cutaneous squamous cell carcinomas in cells with wild-type B-RAF because of paradoxical activation of ERK signaling. Shen et al. showed that this process may be prevented by stimulating AMPK, another kinase that inhibited B-RAF. AMPK is activated in response to signals that indicate a reduction in cellular energy status, and drugs that activate this kinase are used to treat diabetes. Exposure of cells with wild-type B-RAF (mouse embryo fibroblasts, a human keratinocyte cell line, or melanoma cells) to AMPK activators (AICAR, phenformin, or A-769662) reduced phosphorylation (an indicator of activation) of ERK. Mass spectrometry analysis indicated that AICAR treatment of COS-7 cells increased the phosphorylation of tagged B-RAF at Ser729, which was confirmed with endogenous proteins using phosphorylation-specific antibodies. Phosphorylated Ser729 is a recognition site in B-RAF for 14-3-3, a phosphorylation-dependent binding site protein. Braf-null mouse embryo fibroblasts expressing S729A B-RAF failed to exhibit reduced ERK phosphorylation in response to AICAR. In vitro assays confirmed that AMPK phosphorylated this site on B-RAF but also showed that phosphorylation did not reduce B-RAF catalytic activity. Instead, AICAR promoted the coimmunoprecipitation of B-RAF and 14-3-3 and reduced the coimmunoprecipitation of B-RAF and its scaffold KSR, which promotes B-RAF signaling. The S729A mutant also exhibited reduced binding to C-RAF, even in the absence of AMPK activators, suggesting that this site may play a role in the dimerization of the two kinases. When expressed in a keratinocyte cell line, the S729A mutant increased the rate of proliferation, an effect that was not attenuated by AICAR, in contrast to cells overexpressing wild-type B-RAF. Pretreatment of a keratinocyte cell line (having wild-type B-RAF) with AICAR attenuated the induction of ERK phosphorylation stimulated by a subsequent exposure to the B-RAF inhibitor PLX4032. Phenformin administered to mice along with PLX4720 also attenuated epidermal hyperplasia and the abundance of phosphorylated ERK in skin triggered by PLX4720. Thus, drugs currently in use to treat diabetes may serve as an adjunct to chemotherapy targeting mutant B-RAF and prevent unwanted side effects of treatment (see Martin and Marais for commentary).

C.-H. Shen, P. Yuan, R. Perez-Lorenzo, Y. Zhang, S. X. Lee, Y. Ou, J. M. Asara, L. C. Cantley, B. Zheng, Phosphorylation of BRAF by AMPK impairs BRAF-KSR1 association and cell proliferation. Mol. Cell 52, 161–172 (2013). [PubMed]

M. Martin, R. Marais, Braking BRAF: AMPK leaves ERK stranded in the desert. Mol. Cell 52, 155–156 (2013). [Online Journal]

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