Subunits of the Snf1 Kinase Heterotrimer Show Interdependence for Association and Activity

Karin Elbing,Eric M Rubenstein,Rhonda R Mccartney,Martin C Schmidt

doi:10.1074/jbc.m603811200

Karin Elbing, Eric M Rubenstein + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m603811200

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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.

Highlights

Aerobic growth and for fermentation of alternative carbon sources
Whereas ␤ may hold the sucrose non-fermenting-1 (Snf1)/AMPactivated protein kinase (AMPK) heterotrimer together, it is the reversible interactions of the ␣- and ␥-subunits that regulate the enzymatic activity of the kinase domain
An additional layer of regulation of the Snf1/AMPK complex comes from the absolute requirement of the Snf1/AMPK kinases to be phosphorylated on their activation loops by a distinct upstream kinase

Summary

EXPERIMENTAL PROCEDURES

TAP Purifications—For protein purification of tandem affinity purification (TAP)-tagged Sak[1], Sak1-D277A, Snf[1], and Snf1-KD, cells were grown in synthetic complete media containing sucrose (for Snfϩ strains) or glucose (for SnfϪ strains) as the carbon source and harvested in the mid-logarithmic phase. Protein Binding Assays—Lysates from E. coli expressing GST or GST-tagged Snf1-KD, Gal[83] and Snf[4] were bound to glutathione beads (20 ␮l) and washed once with 1 ml NETN and twice with 1 ml of PBS (135 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) containing protease inhibitors (0.5 ␮g/ml leupeptin, 5 ␮g/ml aprotinin, 0.5 ␮g/ml chymostatin 0.5 ␮g/ml pepstatin A, 17 ␮g/ml PMSF). Yeast cells expressing V5-tagged Sak[1] were lysed with glass beads in PBS buffer containing protease inhibitors (1 mM benzamidine, 0.1 mM PMSF, 5 ␮g/ml aprotinin). Size standards used to calibrate the column were bovine thyroglobulin (670 kDa), rabbit muscle aldolase (158 kDa), and chicken ovalbumin (43 kDa)

RESULTS

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DISCUSSION