Subunits of the Snf1 Kinase Heterotrimer Show Interdependence for Association and Activity.
From: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
The Journal of biological chemistry
- Publish Date: Sep 2006
- ISSN: 0021-9258
- Volume: 281
- Issue: 36
- Pages: 26170-80
- Medium: Print
- Language: English
- Citation (JAMA): Elbing Karin, Rubenstein Eric M, McCartney Rhonda R, et al. Subunits of the Snf1 Kinase Heterotrimer Show Interdependence for Association and Activity.. J. Biol. Chem. Sep 2006;281:26170-80
Abstract
The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.
Mesh Headings (Keywords): Animals, DNA-Binding Proteins, Enzyme Activation, Glucose, Multiprotein Complexes, Protein Structure, Quaternary, Protein Subunits, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Check for Full Text / PubMed Unique Identifier (PMID): 16847059
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