Abstract
Emerging evidence has shown that cellular energy metabolism is regulated by the AMPK and MLK3-JNK signaling pathways, but the functional link between them remains to be determined. The present study aimed to explore the crosstalk between MLK3 and AMPK. We found that both JNK and AMPK were phosphorylated at their activation sites by TNF-α, Anisomycin, H2O2 and sorbitol. Interestingly, sorbitol stimulated phosphorylation of AMPK at T172 in LKB1-deficient cells. Following the screening of more than 100 kinases, we identified that MLK3 induced phosphorylation of AMPK at T172. Our in vitro analysis further revealed that MLK3-mediated phosphorylation of AMPK at T172 was independent of AMP, but addition of AMP caused a mobility shift of AMPK, an indication of autophosphorylation, suggesting that AMP binding and phosphorylation of T172 leads to maximal activation of AMPK. GST-pull down assays showed a direct interaction between AMPKα1 subunit and MLK3. Altogether, our results indicate that MLK3 serves as a common upstream kinase of AMPK and JNK and functions as a direct upstream kinase for AMPK independent of LKB1.
Highlights
There are several mitogen-activated-protein kinases (MAPKs) pathways consisting of three tiers of kinases, MAPK, MAPK kinase (MAP2K), and MAPK kinase (MAP3K)
TNF-α, Anisomycin, H2O2 and sorbitol were able to activate both AMPK and JNK (Fig 2), suggesting there might be a crosstalk between AMPK and MLK3-JNK pathways
Our GST-pull down assays showed a direct interaction between AMPKα1 subunit and MLK3
Summary
There are several mitogen-activated-protein kinases (MAPKs) pathways consisting of three tiers of kinases, MAPK, MAPK kinase (MAP2K), and MAPK kinase (MAP3K). When triggered by extracellular and intracellular signals, each is consecutively phosphorylated and activated by its upstream component, leading to an amplification of signaling cascade [1]. MAPK cascades are involved in diverse cellular activities, including mitosis, programmed cell death, motility and metabolism [2]. Substrates for MAPKs include transcription factors, phospholipases, protein kinases, cytoskeleton-associated proteins and membrane receptors. The mixed-lineage kinases (MLKs) are a family of serine/threonine protein kinases, and the catalytic domain of MLK3 resembles both serine and threonine kinase and tyrosine kinase [3]. MLK belongs to MAP3K that contains four isoforms, MLK1, 2, 3 and 4, all of which encompass an amino-terminal SRC-Homology-3 (SH3) domain, a kinase domain, a leucine-zipper region
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