Department of Molecular Genetics and Biochemistry
University of Pittsburgh School of Medicine
Pittsburgh, PA 15261
| The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic α subunit and two non-catalytic subunits, β and γ. The β subunit is thought to hold the complex together and control subcellular localization whereas the γ subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the α 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 β and γ 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 β subunits do not contain any γ subunit, indicating that the Snf1 kinase does not form a stable αγ 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 β and γ subunits could stimulate the kinase activity of the full-length α subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex. |