Phosphorylation of LKB1 at serine 428 by protein kinase C-zeta is required for metformin-enhanced activation of the AMP-activated protein kinase in endothelial cells

Abstract

Background: Metformin, one of most commonly used antidiabetes drugs, is reported to exert its therapeutic effects by activating AMP-activated protein kinase (AMPK); however, the mechanism by which metformin activates AMPK is poorly defined. The objective of the present study was to determine how metformin activates AMPK in endothelial cells.

Methods and results: Exposure of human umbilical vein endothelial cells or bovine aortic endothelial cells to metformin significantly increased AMPK activity and the phosphorylation of both AMPK at Thr172 and LKB1 at Ser428, an AMPK kinase, which was paralleled by increased activation of protein kinase C (PKC)-zeta, as evidenced by increased activity, phosphorylation (Thr410/403), and nuclear translocation of PKC-zeta. Consistently, either pharmacological or genetic inhibition of PKC-zeta ablated metformin-enhanced phosphorylation of both AMPK-Thr172 and LKB1-Ser428, suggesting that PKC-zeta might act as an upstream kinase for LKB1. Furthermore, adenoviral overexpression of LKB1 kinase-dead mutants abolished but LKB1 wild-type overexpression enhanced the effects of metformin on AMPK in bovine aortic endothelial cells. In addition, metformin increased the phosphorylation and nuclear export of LKB1 into the cytosols as well as the association of AMPK with LKB1 in bovine aortic endothelial cells. Similarly, overexpression of LKB1 wild-type but not LKB1 S428A mutants (serine replaced by alanine) restored the effects of metformin on AMPK in LKB1-deficient HeLa-S3 cells, suggesting that Ser428 phosphorylation of LKB1 is required for metformin-enhanced AMPK activation. Moreover, LKB1 S428A, like kinase-dead LKB1 D194A, abolished metformin-enhanced LKB1 translocation as well as the association of LKB1 with AMPK in HeLa-S3 cells. Finally, inhibition of PKC-zeta abolished metformin-enhanced coimmunoprecipitation of LKB1 with both AMPKalpha1 and AMPKalpha2.

Conclusions: We conclude that PKC-zeta phosphorylates LKB1 at Ser428, resulting in LKB1 nuclear export and hence AMPK activation.

Figure 1

Inhibition of PKC-ζ attenuates metformin-enhanced AMPK activity. Confluent BAEC or HUVEC, with or without adenovirus infection, were preincubated with PKC-ζ-PS for 30 minutes before being exposed to metformin. After treatment, the cells were lysaed and extracted. Both phosphorylated AMPK-Thr172 and ACC-Ser79 were detected in Western blots by using specific antibodies, as described in Methods. A, Metformin (Met) activates AMPK in BAEC and HUVEC. The blot is a representative of 6 blots obtained from 6 individual experiments. Con indicates control. B and C, Selective inhibition of PKC-ζ attenuated metformin-enhanced AMPK activity (n=6). ♣P<0.05 (control [Con] vs metformin treated), †P<0.05 (metformin vs metformin plus PKC-ζ-PS). D, PKC-ζ-PS dose-dependently inhibits metformin-enhanced AMPK activity in BAEC (n=5). ♣P<0.05 (control vs metformin), †P<0.05 (metformin vs metformin plus PKC-ζ-PS). E and F, Genetic inhibition of PKC-ζ with PKC-ζ-DN, but not the adenovi-rus encoding GFP, attenuated metformin-enhanced phosphorylation of AMPK-Thr172 and ACC-Ser79 in BAEC. The blot is representative of 5 blots from 5 individual experiments (n=5). ♣P<0.05 (compared with control), †P<0.05 (metformin-treated vs metformin plus adenovirus). G, AMPK activity was assayed as described in Methods (n=4). ♣P<0.05 (compared with control), †P<0.05 (metformin-treated vs metformin plus adenovirus).

Figure 2

Metformin increases PKC-ζ phosphorylation and the translocation of PKC-ζ from cytosol to the membrane. Confluent BAEC were exposed to metformin (1 mmol/L, 1 hour), and the translocation of PKC-ζ and PKC-ζ phosphorylation was assayed as described in Methods. A, Metformin (Met) increased the phosphorylation of PKC-ζ in BAEC. The blot is a representative of 3 blots obtained from 3 independent experiments. Con indicates control. B, PKC-ζ activity was determined as described in Methods (n=4). ♣P<0.05 (compared with control [Con]), †P<0.05 (metformin-treated vs metformin plus adenovirus), #P<0.05 (PKC-ζ-WT vs metformin plus WT adenovirus). C and D, Metformin increases the translocation of PKC-ζ to the membrane (n=5). ♣P<0.05 (control vs metformin-treated). E and F, Metformin increases the translocation of PKC-ζ from cytosol into the nucleus. The blot is a representative of 3 blots from 3 individual experiments (n=5). ♣P<0.05 (control vs metformin-treated). G, Analysis of the purity of subcellular fractions. The subcellular fractions were prepared as described in Methods. Marker enzymes were detected by Western blot with the use of specific antibodies.

Figure 3

Neither PKA nor RSK is involved in metformin-enhanced AMPK activation in BAEC. A and B, Confluent BAEC were pretreated with protein kinase inhibitors for 30 minutes followed by treatment with metformin (1 mmol/L) for 1 hour, the cells were lysed, and phosphorylation of AMPK and LKB1 was detected by Western blot. The blot is a representative of blots from 3 different experiments. ♣P<0.05 compared with respective control (Con). C, BAEC were treated with metformin (Met) (1 mmol/L) for 1 hour, cell lysate was prepared, and the phosphorylation of PKC-ζ, PKAc, and RSK3 was detected by Western blot with the use of specific antibodies. The blot is a representative of 3 blots obtained from 3 individual experiments. D, LKB1 was immunoprecipitated, and PKC-ζ, PKAc, and RSK3 were detected by Western blotting. The blot is representative of blots from 3 different experiments. E, Confluent BAECs were transfected with PKC-ζ-DN and PKC-ζ-WT adenovirus for 48 hours and treated with metformin for 1 hour. The cells were lysed and analyzed by Western blotting. The blot is a representative of 3 blots obtained from 3 individual experiments.

Figure 4

PKC-ζ–dependent LKB1 phosphorylation of LKB1 at serine 428. A and B, Metformin-activated AMPK is LKB1 dependent in BAEC. Adenoviral overexpression of the kinase-dead LKB1 mutant blocks metformin-induced AMPK activation, whereas LKB1 WT overexpression enhanced the effect of LKB1 in BAEC. The blot is a representative of 5 blots from 5 independent experiments. C and D, Metformin-enhanced LKB1 phosphorylation at serine 428 is PKC-ζ dependent. The blot is a representative of 5 blots from 5 independent experiments (n=5). ♣P<0.05 (control [Con] vs metformin), †P<0.05 (metformin vs metformin plus PKC-ζ adenovirus). E, LKB1 activity was measured as described in Methods (n=4). F and G, Time course of PKC-ζ, LKB1, and AMPK phosphorylation. BAEC were treated with metformin (1 mmol/L), and the phosphorylation of PKC-ζ, LKB1, and AMPK was detected at the indicated time by Western blot analysis. The blot is a representative of 3 blots from 3 individual experiments. *♣†P<0.05 compared with respective controls.

Figure 5

Metformin increases the translocation of LKB1 from nucleus into cytosol in HUVEC. A, Immunocytochemical staining of translocation of LKB1 from nucleus to the cytosols caused by metformin in HUVEC. B and C, Metformin (Met) also increases the amount of LKB1 in the cytosol while it decreases LKB1 in the nuclei in HUVEC. The blot is a representative of 5 blots obtained from 5 independent experiments. Con indicates control. D and E, Metformin increases the serine 428 phosphorylation of LKB1 in both nucleus and cytosol. The blot is a representative of 3 blots obtained from 3 independent experiments.

Figure 6

The phosphorylation of LKB1-Ser428 is required for metformin-enhanced translocation of LKB1 and AMPK activation. A, Immunocytochemical staining of metformin (Met)-enhanced LKB1 translocation in A549 cells. The cells were transfected with indicated plasmids, as described in Methods. LKB1 was detected by using a mouse anti-His Tag antibody. The image with Lac-Z was omitted because A549 cells transfected with Lac-Z did not react with the antibody. Con indicates control. B, Western blot detection of LKB1 in A549 cells. The blot is a representative of 5 blots from 5 independent experiments (n=5). ♣P<0.05 (WT vs WT plus metformin), †P<0.05 (WT plus metformin vs WT or S428A plus metformin).

Figure 7

Ser428 phosphorylation of LKB1 is required for metformin-enhanced AMPK activation and coimmunoprecipitation of LKB1 with AMPK. A and B, Metformin activates AMPK in HeLa-S3 overexpressing LKB1-WT but not LKB1-Ser428A mutants. After being transfected with LKB1-WT or kinase-dead LKB1 mutants or the LKB1-Ser428A mutant, HeLa-S3 cells were exposed to metformin (1 mmol/L, 1 hour). AMPK activation was monitored in Western blots with the use of specific antibodies. The blot is a representative of 5 blots from 5 independent experiments. C, Ser428 phosphorylation of LKB1 is required for ONOO⁻-enhanced AMPK activation. After being transfected with LKB1-WT or LKB1-Ser428A (Ser428 was mutated into alanine, loss of function) or the LKB1-Ser428D (Ser428 replaced by aspartic acid, phosphorylation mimicking), HeLa-S3 cells were exposed to ONOO⁻ (100 μmol/L). AMPK activation was monitored by Western blotting with the use of specific antibodies. The blot is a representative of 5 blots from 5 independent experiments. D, LKB1 activity in LKB1 WT or LKB1-Ser431A mutants. E, Metformin increases the association of LKB1 and AMPK. LKB1 was immunoprecipitated from BAEC, and AMPK was detected in Western blots. Inhibition of PKC-ζ attenuated metformin-enhanced association of LKB1 with AMPK. The blot is a representative of 5 blots from 5 independent experiments. F, Mutation of Ser428 of LKB1 into alanine (S428A) abolishes metformin-enhanced coimmunoprecipitation of LKB1 with AMPK. Metformin increased the coimmunoprecipitation of LKB1 with LKB1 WT but not with LKB1 mutant. The blot is a representative of 3 blots from 3 individual experiments.

Figure 8

LKB1 nuclear export is required for metformin-enhanced AMPK activation. In the resting state, LKB1 is mainly located in the nucleus, whereas LKB1 activity required that adaptor proteins MO25 and STRAD as well as AMPK-α1 (the major isoform of AMPK catalytic units in endothelial cells) be located in the cytosol. Metformin (or ONOO⁻) activates PI-3 kinase, resulting in PDK-1/2 phosphorylation. PDK-1/2 phosphorylates PKC-ζ, resulting in the translocation of the latter into nucleus as well as cytoplasmic membrane. In the nucleus, activated PKC-ζ phosphorylates LKB1 at serine 428 and likely other sites. Phosphorylated LKB1 is exported from the nucleus via an unknown mechanism into the cytosol. LKB1 is subsequently bound to the preexisting adaptors proteins (STRAD and MO25), which recruit and phosphorylate AMPK at Thr172. AMPK is likely also activated by other stimuli, which either activate PKC-ζ or increase LKB1-Ser428 phosphorylation (PKA or RSK90). Overall, our results suggest that the PI-3 kinase-PDK1/2-PKC-ζ pathway plays a critical role in metformin-enhanced AMPK activation.