Identification of Phosphorylation Sites in AMP-activated Protein Kinase (AMPK) for Upstream AMPK Kinases and Study of Their Roles by Site-directed Mutagenesis
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Date
2003-08-01
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Journal Article
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yes
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Abstract
Bacterially expressed heterotrimeric (α₁, β₁, and γ₁) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the α-subunit, viz. Thr²⁵⁸ and Ser⁴⁸⁵ (Ser⁴⁹¹ in the α₂-subunit) by mass spectrometry, in addition to the previously characterized Thr¹⁷² site. Also, autophosphorylation sites in the β₁-subunit were identified as Ser⁹⁶, Ser¹⁰¹, and Ser¹⁰⁸. Mutagenesis of Thr¹⁷², Thr²⁵⁸, and Ser⁴⁸⁵ to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr¹⁷² was involved in AMPK activation, whereas Thr²⁵⁸ and Ser⁴⁸⁵ were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the α-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser⁴⁸⁵ phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition.
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Journal / series
Volume
278 (31)
Pages / Article No.
28434 - 28442
Publisher
American Society for Biochemistry and Molecular Biology