Fig 1.
In response to extracelluar stimuli, MLK3 is dimerized and activated by Cdc42/Rac [5, 8]. Once activated, MLK3 will activate multipe MAP2Ks, which in turn activate the downstream MAPKs by phosphorylation, such as ERK1/2, JNK, or p38 MAPK. The activated MAPKs will further participate in regulating various cellular processes via transcription factors.
Fig 2.
Activation of AMPK and JNK/MAPK by stresses and ligands.
A: A stable A549 cell line was established by infection of lentivirus carrying LKB1 cDNA. LKB1 expression was examined by Western blot. B: A549 cells and stable A549-LKB1 cells were treated with IL6 (10 ng/ml), TNF-α (10 ng/ml), Adiponectin (10 μg/ml), Anisomycin (10 μg/ml), H2O2 (10 mM) and sorbitol (2.5 mM) for 30 min, or AICAR (1mM) for 2 h. Total proteins were extracted and blotted with antibodies as indicated.
Fig 3.
MLK3 phophorylated AMPKα at T172.
A: HEK293 cells were transfected with empty control, MLK3 or LKB1 vectors. Total proteins were extracted for immunoblotting of pAMPKα-T172 and AMPKα. B: Recombinant AMPK and MLK3 were respectively expressed in HEK293T cells and purified by GSH. In vitro kinase assays were performed as indicated in the presence or absence of AMP. The phosphorylation of AMPK at T172 was determined by western blot analysis.
Fig 4.
MLK3 physically interacted with AMPKα1.
A: HEK293T cells were transfected with GST-MLK3 or GST-LKB1, and then MLK3 and LKB1 were pulled down with GSH beads and blotted with anti-GST antibody. B: HEK293T cells were transfected with GST empty vector, GST-MLK3 or GST-LKB1, and then recombinant proteins were pulled down with GSH beads and blotted with anti-AMPKα, anti-AMPKα1, as well as anti-AMPKα2. C: GST-MLK3 or GST was co-expressed either with Myc-AMPKα1 or Myc-AMPKα2 in HEK293T cells. Immunoblotting was performed on GSH-pulldown and crude extracts with antibodies against GST and Myc, respectively.
Fig 5.
Model of crosstalk between MLK3 and AMPK in stress responses.