Department of Molecular Genetics and Biochemistry
University of Pittsburgh School of Medicine
Pittsburgh, Pennsylvania 15261
| Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. Neither Sak1, Tos3 nor Elm1 appear to require the presence of additional subunits for activity. One of the Snf1-activating kinases, Sak1, forms a complex with Snf1 since the two proteins co-purify with each other and co-elute in size exclusion chromatography. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of beta and gamma subunits of the Snf1 heterotrimer. Finally, we show that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its beta and gamma subunits. |