A pivotal role for endogenous TGF-beta-activated kinase-1 in the LKB1/AMP-activated protein kinase energy-sensor pathway

Abstract

TGF-beta-activated kinase-1 (TAK1), also known as MAPKK kinase-7 (MAP3K7), is a candidate effector of multiple circuits in cardiac biology and disease. Here, we show that inhibition of TAK1 in mice by a cardiac-specific dominant-negative mutation evokes electrophysiological and biochemical properties reminiscent of human Wolff-Parkinson-White syndrome, arising from mutations in AMP-activated protein kinase (AMPK), most notably, accelerated atrioventricular conduction and impaired AMPK activation. To test conclusively the biochemical connection from TAK1 to AMPK suggested by this phenotype, we disrupted TAK1 in mouse embryos and embryonic fibroblasts by Cre-mediated recombination. In TAK1-null embryos, the activating phosphorylation of AMPK at T172 was blocked, accompanied by defective AMPK activity. However, loss of endogenous TAK1 causes midgestation lethality, with defective yolk sac and intraembryonic vasculature. To preclude confounding lethal defects, we acutely ablated floxed TAK1 in culture by viral delivery of Cre. In culture, endogenous TAK1 was activated by oligomycin, the antidiabetic drug metformin, 5-aminoimidazole-4-carboxamide riboside (AICAR), and ischemia, well established triggers of AMPK activity. Loss of TAK1 in culture blocked T172 phosphorylation induced by all three agents, interfered with AMPK activation, impaired phosphorylation of the endogenous AMPK substrate acetyl CoA carboxylase, and also interfered with activation of the AMPK kinase LKB1. Thus, by disrupting the endogenous TAK1 locus, we prove a pivotal role for TAK1 in the LKB1/AMPK signaling axis, an essential governor of cell metabolism.

Fig. 1.

Cardiac-specific inhibition of TAK1 causes accelerated AV conduction. (A) The Cre-dependent dnTAK1 gene (stop-dnTAK). CAG, CMV enhancer + chicken β-actin promoter; CAT, chloramphenicol acetyltransferase; pA, polyadenylation signal. (B) Transgene expression was contingent on stop-dnTAK plus αMyHC-Cre (PCR, above) and blocked activation of JNK and p38 [Western blot (WB), below]. (C) Lead II ECGs and simultaneous intracardiac His bundle electrograms (HBE), showing accelerated atrial-to-His bundle conduction (abbreviated A–H interval).

Fig. 2.

dnTAK1 prevents the activating phosphorylation of AMPK. (A) Specificity of the AMPKα1 and -α2 antibodies by Western blotting, using transfected 293 cells. (B and C) Transgenic mouse myocardium (day 6). (B) Cre/stop-dnTAK1 inhibits AMPKα T-loop phosphorylation (Left), phosphorylation of the AMPK substrate acetyl-CoA carboxylase 1 (Left), and AMPK activity (Center Right). (C) Cre/stop-dnTAK1 inhibits LKB1 activity. (D–G) Viral gene delivery to cultured cardiomyocytes. (D) dnTAK1 blocks oligomycin-induced AMPKα phosphorylation. (E) dnTAK1 prevents metformin-induced AMPKα phosphorylation. (F) dnTAK1 inhibits activation of the AMPK kinase, LKB1, by MO25α and STRADα. (G) MAP4K4 induces AMPKα phosphorylation by TAK1. n ≥ 3; P < 0.05.

Fig. 3.

AMPK-activating signals activate endogenous TAK1. Cardiac myocytes were treated with oligomycin, 500 nM (A); metformin, 2.5 mM (B); and 5-aminoimidazole-4-carboxamide riboside, 1 mM (C); and isolated perfused rat hearts were subjected to ischemic injury (D). TAK1 activation is shown by the immune complex kinase assay, and TAK1 protein levels are indicated by Western blotting. n ≥ 3; P < 0.05.

Fig. 4.

Endogenous TAK1 is essential for normal AMPK activation. (A and B) Mouse embryos (embryonic day 9). (A) Loss of TAK1 disrupts AMPKα phosphorylation. No change was seen in AMPK subunit expression or levels of LKB1. (B) Loss of TAK1 disrupts inhibits AMPKα1, AMPKα2, and LKB1 activities. (C–F) Viral delivery of Cre to mouse embryo fibroblasts. (C–E) Deleting TAK1 disrupts agonist-induced phosphorylation of AMPKα, AMPK activity, and phosphorylation of acetyl-CoA carboxylase. (F) Deleting TAK1 reduced by >80% the activation of LKB1 by coexpressed MO25α and STRADα. (G) Schematic model coupling TAK1 to AMPK phosphorylation and activity. n ≥ 3; P < 0.05.