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AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit.

Publication ,  Journal Article
Hamilton, SR; O'Donnell, JB; Hammet, A; Stapleton, D; Habinowski, SA; Means, AR; Kemp, BE; Witters, LA
Published in: Biochem Biophys Res Commun
May 10, 2002

The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress. It consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, and is regulated both by the allosteric action of AMP and by phosphorylation of the alpha and beta subunits catalyzed by AMPKK(s) and autophosphorylation. The Thr172 site on the alpha subunit has been previously characterized as an activating phosphorylation site. Using bacterially expressed AMPK alpha1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in alpha subunit regulation. Recombinant alpha1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6x His fusion proteins and purified to homogeneity by Ni(2+) chromatography. Both wild-type alpha1(1-312) and alpha1(1-312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKbeta. The corresponding AMPKalpha1(1-392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive. In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK. Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant alpha1(1-312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active.

Duke Scholars

Published In

Biochem Biophys Res Commun

DOI

ISSN

0006-291X

Publication Date

May 10, 2002

Volume

293

Issue

3

Start / End Page

892 / 898

Location

United States

Related Subject Headings

  • Tumor Cells, Cultured
  • Ribonucleotides
  • Recombinant Fusion Proteins
  • Protein Subunits
  • Protein Kinases
  • Phosphothreonine
  • Phosphorylation
  • Glucose
  • Biochemistry & Molecular Biology
  • Animals
 

Citation

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Hamilton, S. R., O’Donnell, J. B., Hammet, A., Stapleton, D., Habinowski, S. A., Means, A. R., … Witters, L. A. (2002). AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit. Biochem Biophys Res Commun, 293(3), 892–898. https://doi.org/10.1016/S0006-291X(02)00312-1
Hamilton, Stephen R., John B. O’Donnell, Andrew Hammet, David Stapleton, Susan A. Habinowski, Anthony R. Means, Bruce E. Kemp, and Lee A. Witters. “AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit.Biochem Biophys Res Commun 293, no. 3 (May 10, 2002): 892–98. https://doi.org/10.1016/S0006-291X(02)00312-1.
Hamilton SR, O’Donnell JB, Hammet A, Stapleton D, Habinowski SA, Means AR, et al. AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit. Biochem Biophys Res Commun. 2002 May 10;293(3):892–8.
Hamilton, Stephen R., et al. “AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit.Biochem Biophys Res Commun, vol. 293, no. 3, May 2002, pp. 892–98. Pubmed, doi:10.1016/S0006-291X(02)00312-1.
Hamilton SR, O’Donnell JB, Hammet A, Stapleton D, Habinowski SA, Means AR, Kemp BE, Witters LA. AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit. Biochem Biophys Res Commun. 2002 May 10;293(3):892–898.
Journal cover image

Published In

Biochem Biophys Res Commun

DOI

ISSN

0006-291X

Publication Date

May 10, 2002

Volume

293

Issue

3

Start / End Page

892 / 898

Location

United States

Related Subject Headings

  • Tumor Cells, Cultured
  • Ribonucleotides
  • Recombinant Fusion Proteins
  • Protein Subunits
  • Protein Kinases
  • Phosphothreonine
  • Phosphorylation
  • Glucose
  • Biochemistry & Molecular Biology
  • Animals