Limits...
Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5.

Yan Y, Tsukamoto O, Nakano A, Kato H, Kioka H, Ito N, Higo S, Yamazaki S, Shintani Y, Matsuoka K, Liao Y, Asanuma H, Asakura M, Takafuji K, Minamino T, Asano Y, Kitakaze M, Takashima S - Nat Commun (2015)

Bottom Line: Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation.Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery.Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT
Augmented AMP-activated protein kinase (AMPK) activity inhibits cell migration, possibly contributing to the clinical benefits of chemical AMPK activators in preventing atherosclerosis, vascular remodelling and cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we identify PDZ and LIM domain 5 (Pdlim5) as a novel AMPK substrate and show that it plays a critical role in the inhibition of cell migration. AMPK directly phosphorylates Pdlim5 at Ser177. Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation. Consistent with this observation, S177D-Pdlim5 suppresses Rac1 activity at the cell periphery and displaces the Arp2/3 complex from the leading edge. Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery. Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway.

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AMPK is recruited onto F-actin by directly binding the LIM domain of Pdlim5.(a) HEK293T cells were co-transfected with V5-tagged AMPKα and FLAG-tagged Pdlim5 (WT, ΔPDZ or ΔLIM domain). TCLs were immunoprecipitated by FLAG or V5 and then immunoblotted with the indicated antibodies. (b) F-actin binding assay of AMPK. AMPK was mixed with a fixed amount of F-actin in the presence or absence of α-actinin and GST-Pdlim5, incubated for 1 h at 24 °C and then centrifuged at 150,000 g for 1.5 h at 24 °C, to pellet the F-actin polymer and associated proteins. A sample of the pellet (P) and supernatant (S) were analysed by immunoblotting for the indicated antibodies.
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f9: AMPK is recruited onto F-actin by directly binding the LIM domain of Pdlim5.(a) HEK293T cells were co-transfected with V5-tagged AMPKα and FLAG-tagged Pdlim5 (WT, ΔPDZ or ΔLIM domain). TCLs were immunoprecipitated by FLAG or V5 and then immunoblotted with the indicated antibodies. (b) F-actin binding assay of AMPK. AMPK was mixed with a fixed amount of F-actin in the presence or absence of α-actinin and GST-Pdlim5, incubated for 1 h at 24 °C and then centrifuged at 150,000 g for 1.5 h at 24 °C, to pellet the F-actin polymer and associated proteins. A sample of the pellet (P) and supernatant (S) were analysed by immunoblotting for the indicated antibodies.

Mentions: To investigate how AMPK signalling is transmitted to peripheral actin filaments, we examined the physical link between actin filaments, Pdlim5 and AMPK. Immunoprecipitation/immunoblotting of HEK293T cells co-transfected with V5-tagged AMPKα and FLAG-tagged Pdlim5 (WT, ΔPDZ or ΔLIM) demonstrated that AMPK bound to Pdlim5 through the LIM domain (Fig. 9a). Next, we performed an F-actin-binding assay, in which F-actin and its binding proteins are found in the pellet fraction, to determine whether Pdlim5 promotes the recruitment of AMPK onto actin filaments. In the absence of Pdlim5, AMPK was found exclusively in the supernatant (Fig. 9b). However, in the presence of Pdlim5, AMPK shifted from the supernatant to the pellets and this shift was greatly stimulated by the presence of α-actinin (Fig. 9b). These findings indicate that Pdlim5 binds AMPK directly and promotes the recruitment of AMPK onto F-actin, a process mediated by α-actinin.


Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5.

Yan Y, Tsukamoto O, Nakano A, Kato H, Kioka H, Ito N, Higo S, Yamazaki S, Shintani Y, Matsuoka K, Liao Y, Asanuma H, Asakura M, Takafuji K, Minamino T, Asano Y, Kitakaze M, Takashima S - Nat Commun (2015)

AMPK is recruited onto F-actin by directly binding the LIM domain of Pdlim5.(a) HEK293T cells were co-transfected with V5-tagged AMPKα and FLAG-tagged Pdlim5 (WT, ΔPDZ or ΔLIM domain). TCLs were immunoprecipitated by FLAG or V5 and then immunoblotted with the indicated antibodies. (b) F-actin binding assay of AMPK. AMPK was mixed with a fixed amount of F-actin in the presence or absence of α-actinin and GST-Pdlim5, incubated for 1 h at 24 °C and then centrifuged at 150,000 g for 1.5 h at 24 °C, to pellet the F-actin polymer and associated proteins. A sample of the pellet (P) and supernatant (S) were analysed by immunoblotting for the indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317497&req=5

f9: AMPK is recruited onto F-actin by directly binding the LIM domain of Pdlim5.(a) HEK293T cells were co-transfected with V5-tagged AMPKα and FLAG-tagged Pdlim5 (WT, ΔPDZ or ΔLIM domain). TCLs were immunoprecipitated by FLAG or V5 and then immunoblotted with the indicated antibodies. (b) F-actin binding assay of AMPK. AMPK was mixed with a fixed amount of F-actin in the presence or absence of α-actinin and GST-Pdlim5, incubated for 1 h at 24 °C and then centrifuged at 150,000 g for 1.5 h at 24 °C, to pellet the F-actin polymer and associated proteins. A sample of the pellet (P) and supernatant (S) were analysed by immunoblotting for the indicated antibodies.
Mentions: To investigate how AMPK signalling is transmitted to peripheral actin filaments, we examined the physical link between actin filaments, Pdlim5 and AMPK. Immunoprecipitation/immunoblotting of HEK293T cells co-transfected with V5-tagged AMPKα and FLAG-tagged Pdlim5 (WT, ΔPDZ or ΔLIM) demonstrated that AMPK bound to Pdlim5 through the LIM domain (Fig. 9a). Next, we performed an F-actin-binding assay, in which F-actin and its binding proteins are found in the pellet fraction, to determine whether Pdlim5 promotes the recruitment of AMPK onto actin filaments. In the absence of Pdlim5, AMPK was found exclusively in the supernatant (Fig. 9b). However, in the presence of Pdlim5, AMPK shifted from the supernatant to the pellets and this shift was greatly stimulated by the presence of α-actinin (Fig. 9b). These findings indicate that Pdlim5 binds AMPK directly and promotes the recruitment of AMPK onto F-actin, a process mediated by α-actinin.

Bottom Line: Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation.Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery.Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

ABSTRACT
Augmented AMP-activated protein kinase (AMPK) activity inhibits cell migration, possibly contributing to the clinical benefits of chemical AMPK activators in preventing atherosclerosis, vascular remodelling and cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we identify PDZ and LIM domain 5 (Pdlim5) as a novel AMPK substrate and show that it plays a critical role in the inhibition of cell migration. AMPK directly phosphorylates Pdlim5 at Ser177. Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation. Consistent with this observation, S177D-Pdlim5 suppresses Rac1 activity at the cell periphery and displaces the Arp2/3 complex from the leading edge. Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery. Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway.

Show MeSH
Related in: MedlinePlus