Figure 6.
AMPK phosphorylates TRAK1. (A) MYC-tagged hTRAK1, as well as an empty vector, constitutive active AMPK (CA-AMPK), or dominant negative AMPK (DN-AMPK), was co-expressed in HEK293T cells. MYC-hTRAK1 was then immunoprecipitated using an anti-MYC antibody. Immunoprecipitates and lysates were blotted and then probed for phosphorylated AMPK substrate, anti-MYC, and GAPDH immunoreactivity. (B) Quantification of the phosphorylated AMPK substrate epitope normalized to anti-MYC levels from western blots as in A. n = 5 independent transfections per condition. Note that an outlier with a value of 9.5 AMPK substrate/MYC-TRAK signal was removed from the CA-AMPK treatment column, as well as the statistical analysis (this column thus contains 4 data points). (C) Schematic of the hTRAK1 phosphorylation sites. Mass spectrometry was performed on MYC-hTRAK1 expressed in HEK293T with CA-AMPK or DN-AMPK and then immunoprecipitated using an anti-MYC antibody. Immunoprecipitates were run on an acrylamide gel, and a ∼1 cm2 band of ∼100 KD was excised and used for mass spectrometry. Any site identified in the CA-AMPK samples is marked with a balloon. Magenta balloons are those that match the AMPK consensus sequence. The illustration was made with the IBS 2.0 online illustration tool using the Q9UPV9 UniProt human TRAK1 amino acid sequence (Xie et al., 2022). Predicted coiled-coil domains (CC1, CC2) and the OBD are indicated. (D) MYC-hTRAK1 was expressed from an hSYN1 promoter in cortical neuron cultures that were treated with 4 nM AntA or vehicle for 2 h. Anti-MYC immunoprecipitates and lysates were blotted and probed for phosphorylated AMPK substrate, MYC, and HSP90 immunoreactivity. (E) Quantification of TRAK phosphorylation in immunoprecipitates as in D. n = 3 cortical neuron cultures from three independent animals. (F) Quantification of perimitochondrial actin to assess the effects of a 3-day TRAK1 knockdown. Fibroblasts were cultured in galactose media to increase dependence on ETC function and then treated for 1 h with 40 nM AntA or vehicle. F-actin and mitochondria were visualized using GFP-F-tractin and mito-mRaspberry and expressed either nontargeting control or TRAK1 shRNAs. N = 11–17 cells per repeat from 3 independent animals (G) MYC-hTRAK1 with the indicated mutations of phosphorylation sites or with all 5 sites mutated (quintuple) was expressed in HEK293T cells co-expressing CA-AMPK. Anti-MYC immunoprecipitates and lysates were blotted and probed for phosphorylated AMPK substrate, MYC, and GAPDH immunoreactivity. (H) Quantification of G. n = 4 independent transfections per condition. For all graphs, bars on boxplots show the 10–90th percentile, and error bars on bar graphs show the SEM. The P values for B and H were calculated by performing a blocked one-way ANOVA with Dunnett’s T3 multiple comparison correction. The P values for F were calculated by performing a blocked one-way ANOVA with Tukey’s multiple comparison correction. Select P values are shown. P values for E were calculated by performing two-tailed, unpaired t tests with Welch’s correction. OBD, OGT-binding domain. Source data are available for this figure: SourceData F6.

AMPK phosphorylates TRAK1. (A) MYC-tagged hTRAK1, as well as an empty vector, constitutive active AMPK (CA-AMPK), or dominant negative AMPK (DN-AMPK), was co-expressed in HEK293T cells. MYC-hTRAK1 was then immunoprecipitated using an anti-MYC antibody. Immunoprecipitates and lysates were blotted and then probed for phosphorylated AMPK substrate, anti-MYC, and GAPDH immunoreactivity. (B) Quantification of the phosphorylated AMPK substrate epitope normalized to anti-MYC levels from western blots as in A. n = 5 independent transfections per condition. Note that an outlier with a value of 9.5 AMPK substrate/MYC-TRAK signal was removed from the CA-AMPK treatment column, as well as the statistical analysis (this column thus contains 4 data points). (C) Schematic of the hTRAK1 phosphorylation sites. Mass spectrometry was performed on MYC-hTRAK1 expressed in HEK293T with CA-AMPK or DN-AMPK and then immunoprecipitated using an anti-MYC antibody. Immunoprecipitates were run on an acrylamide gel, and a ∼1 cm2 band of ∼100 KD was excised and used for mass spectrometry. Any site identified in the CA-AMPK samples is marked with a balloon. Magenta balloons are those that match the AMPK consensus sequence. The illustration was made with the IBS 2.0 online illustration tool using the Q9UPV9 UniProt human TRAK1 amino acid sequence (Xie et al., 2022). Predicted coiled-coil domains (CC1, CC2) and the OBD are indicated. (D) MYC-hTRAK1 was expressed from an hSYN1 promoter in cortical neuron cultures that were treated with 4 nM AntA or vehicle for 2 h. Anti-MYC immunoprecipitates and lysates were blotted and probed for phosphorylated AMPK substrate, MYC, and HSP90 immunoreactivity. (E) Quantification of TRAK phosphorylation in immunoprecipitates as in D. n = 3 cortical neuron cultures from three independent animals. (F) Quantification of perimitochondrial actin to assess the effects of a 3-day TRAK1 knockdown. Fibroblasts were cultured in galactose media to increase dependence on ETC function and then treated for 1 h with 40 nM AntA or vehicle. F-actin and mitochondria were visualized using GFP-F-tractin and mito-mRaspberry and expressed either nontargeting control or TRAK1 shRNAs. N = 11–17 cells per repeat from 3 independent animals (G) MYC-hTRAK1 with the indicated mutations of phosphorylation sites or with all 5 sites mutated (quintuple) was expressed in HEK293T cells co-expressing CA-AMPK. Anti-MYC immunoprecipitates and lysates were blotted and probed for phosphorylated AMPK substrate, MYC, and GAPDH immunoreactivity. (H) Quantification of G. n = 4 independent transfections per condition. For all graphs, bars on boxplots show the 10–90th percentile, and error bars on bar graphs show the SEM. The P values for B and H were calculated by performing a blocked one-way ANOVA with Dunnett’s T3 multiple comparison correction. The P values for F were calculated by performing a blocked one-way ANOVA with Tukey’s multiple comparison correction. Select P values are shown. P values for E were calculated by performing two-tailed, unpaired t tests with Welch’s correction. OBD, OGT-binding domain. Source data are available for this figure: SourceData F6.

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