The RAF-MEK-ERK signaling cascade plays a central role in transmitting mitogenic signals to regulate cell proliferation, survival, and differentiation. Among the RAF family kinases, BRAF is frequently mutated in human cancers, particularly melanoma, where the V600E mutation leads to constitutive activation of downstream MEK-ERK signaling. While BRAF inhibitors have shown remarkable clinical efficacy in treating BRAF-mutant melanomas, they paradoxically activate ERK signaling in cells with wild-type BRAF, contributing to the development of cutaneous squamous cell carcinomas (cSCC) in patients. This study identifies AMP-activated protein kinase (AMPK), a key cellular energy sensor, as a direct regulator of BRAF activity through phosphorylation at Ser729. Upon energy stress, AMPK is activated and phosphorylates BRAF at Ser729, which enhances its interaction with 14-3-3 adaptor proteins and disrupts its association with the scaffolding protein KSR1. This disruption impairs BRAF’s ability to form active complexes necessary for efficient MEK-ERK activation. As a result, AMPK-mediated phosphorylation significantly attenuates ERK signaling in keratinocytes and reduces cell proliferation. Importantly, this regulatory mechanism operates specifically in cells expressing wild-type BRAF, as BRAF V600E mutants fail to undergo this phosphorylation due to impaired AMPK activation in these cells. The findings reveal that AMPK functions as a metabolic brake on the RAF-MEK-ERK pathway, preventing excessive mitogenic signaling under low-energy conditions. Furthermore, pharmacological activation of AMPK using agents like phenformin or A-769662 effectively suppresses BRAF inhibitor-induced ERK hyperactivation and epidermal hyperplasia in mouse skin models. These results suggest that co-administration of AMPK activators with BRAF inhibitors may represent a promising strategy to mitigate the risk of cSCC development during targeted therapy. By linking cellular energy status to growth control, this mechanism provides insight into how metabolic stress can naturally limit proliferative responses, offering new avenues for therapeutic intervention in cancer and other proliferative disorders.
Activation of AMPK Suppresses RAF-MEK-ERK Signaling via BRAF Phosphorylation
AMPK serves as a master regulator of cellular energy homeostasis, becoming activated in response to metabolic stress such as glucose deprivation, hypoxia, or increased AMP/ADP levels. Once activated, AMPK orchestrates a broad reprogramming of metabolism to restore ATP balance by stimulating catabolic pathways and inhibiting anabolic processes. Beyond its metabolic roles, emerging evidence indicates that AMPK also regulates cell growth and proliferation. In this study, we demonstrate that AMPK directly modulates the RAF-MEK-ERK pathway by phosphorylating BRAF at Ser729.1,2-Bis(pyridin-4-ylmethylene)hydrazine Biological Activity Using multiple cell models—including CCD1106 keratinocytes, mouse embryonic fibroblasts (MEFs), and C140 melanocytes—we show that treatment with AMPK activators such as AICAR, phenformin, and A-769662 leads to a dose-dependent suppression of ERK phosphorylation. Notably, this effect is abolished in AMPK-null MEFs, confirming that AMPK is essential for this regulation. Mass spectrometry analysis of FLAG-tagged BRAF immunoprecipitated from Cos-7 cells revealed a significant increase in phosphorylation at Ser729 upon AICAR treatment, with minimal detection in untreated controls. Moreover, recombinant AMPK directly phosphorylated BRAF in vitro, and mutation of Ser729 to alanine abrogated this phosphorylation. An antibody specific for pSer729 BRAF confirmed robust phosphorylation in response to AMPK activation in both human keratinocytes and MEFs. These data collectively establish that AMPK is the long-sought kinase responsible for phosphorylating BRAF at Ser729 in vivo. The phosphorylation site lies within a sequence motif consistent with AMPK substrate specificity, further supporting its direct targeting. This finding reveals a previously unknown layer of regulation in the RAF-MEK-ERK cascade, where energy-sensing mechanisms directly influence oncogenic signaling pathways.
AMPK-Mediated Phosphorylation of BRAF Disrupts Scaffolding Complexes and Limits Cell Growth
Upon phosphorylation at Ser729, BRAF exhibits enhanced binding to 14-3-3 proteins, which are critical signaling adaptors involved in subcellular localization and functional modulation of their clients. Our immunoprecipitation experiments demonstrated that AICAR treatment promotes the association between endogenous BRAF and 14-3-3 in wild-type MEFs but not in AMPK-null cells. Similarly, GST-14-3-3 pull-down assays confirmed this interaction in CCD1106 keratinocytes. Importantly, this phosphorylation event selectively disrupts the interaction between BRAF and the scaffold protein KSR1, which is essential for efficient MEK activation. Co-immunoprecipitation studies showed reduced BRAF-KSR1 complex formation following AMPK activation, and this effect was lost when Ser729 was mutated to alanine. Interestingly, while AMPK activation also disrupted BRAF-CRAF heterodimerization, the S729A mutant failed to bind CRAF regardless of AMPK status, suggesting that the hydroxyl group of Ser729 contributes to dimer stability even in the absence of 14-3-3 binding. We further found that high-affinity 14-3-3 binding requires phosphorylation of both Ser729 and Ser365, another known 14-3-3 docking site. Mutation of either site impairs 14-3-3 association and allows sustained ERK activation despite AMPK stimulation. These findings support a model in which AMPK phosphorylation of BRAF induces a conformational change that promotes 14-3-3 binding and sterically hinders interactions with KSR1 and CRAF, thereby dampening the overall signaling output. This mechanism ensures that mitogenic signaling is restrained under energy-deficient conditions, preserving cellular resources for essential survival functions.
AMPK Activation Inhibits Keratinocyte Proliferation and Prevents Skin Hyperplasia Induced by BRAF Inhibitors
Given the central role of RAF-MEK-ERK signaling in driving cell cycle progression and proliferation, we investigated whether AMPK-mediated BRAF phosphorylation affects cellular growth. Stable expression of BRAF S729A mutant in CCD1106 keratinocytes resulted in significantly higher proliferation rates compared to cells expressing wild-type BRAF, indicating that Ser729 phosphorylation acts as a negative regulator of cell growth. Flow cytometry analysis revealed that AICAR treatment induced G2/M phase arrest and S-phase accumulation in wild-type BRAF-expressing cells, whereas the S729A mutant cells were largely resistant to these effects.Alclometasone dipropionate (Standard) Data Sheet Consistent with this, AICAR exerted a stronger inhibitory effect on proliferation in WT BRAF cells than in S729A-expressing cells.PMID:33797671 These results confirm that AMPK-dependent phosphorylation of BRAF at Ser729 plays a crucial role in regulating the cell cycle and proliferation in keratinocytes. More importantly, we tested whether AMPK activation could counteract the adverse effects of BRAF inhibitors. In mouse skin, administration of PLX4720 led to significant epidermal hyperplasia and increased Ki67 staining, indicative of hyperproliferation. However, co-treatment with the AMPK activator phenformin markedly reduced both epidermal thickness and proliferation index. Immunohistochemistry confirmed that phenformin prevented PLX4720-induced phosphorylation of ERK and upregulation of Ki67. Similar protective effects were observed with A-769662. These findings demonstrate that AMPK activation can effectively antagonize the paradoxical activation of ERK signaling caused by BRAF inhibitors in normal tissues, thereby preventing the development of cSCC-like lesions. This highlights the therapeutic potential of combining AMPK activators with BRAF-targeted therapies to improve safety profiles without compromising anti-tumor efficacy.
Mechanistic Insights into AMPK Regulation of BRAF and Implications for Cancer Therapy
This study uncovers a fundamental mechanism by which cellular energy status governs oncogenic signaling through direct post-translational modification of BRAF. The identification of AMPK as the kinase responsible for phosphorylating BRAF at Ser729 resolves a longstanding question about the regulation of this key residue. Unlike other kinases that modulate BRAF activity through feedback loops or allosteric regulation, AMPK acts as a primary controller that integrates metabolic cues into growth decisions. By promoting 14-3-3 binding and disrupting critical scaffolding interactions, AMPK effectively silences BRAF signaling under conditions of energy stress, acting as a physiological safeguard against uncontrolled proliferation. The differential sensitivity of BRAF V600E mutants to this regulation underscores a key vulnerability: these tumors are defective in AMPK activation due to ERK-mediated inhibition of upstream kinases like LKB1, rendering them insensitive to this metabolic checkpoint. This explains why BRAF inhibitors trigger side effects only in normal tissues—where wild-type BRAF remains responsive to AMPK regulation—while failing to affect the tumor itself. Therefore, targeting AMPK in non-cancerous tissues offers a rational approach to prevent drug-induced toxicities. Future therapeutic strategies may involve the use of safe, bioavailable AMPK activators such as phenformin or metformin as adjuncts to BRAF inhibitors. Such combinations could maintain anti-tumor efficacy while minimizing risks of secondary malignancies, representing a major advancement in precision oncology.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com