Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae.
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. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. 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 showed 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.
Elbing
Karin
K
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
McCartney
Rhonda R
RR
Schmidt
Martin C
MC
D047390:Casein Kinase II / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D005947:Glucose / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity
C500410:Hym1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C508938:Elm2 protein, S cerevisiae; C071570:SNF1-related protein kinases; C094326:PRK1 protein, S cerevisiae; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; C502450:TOS3 protein, S cerevisiae; D005947:Glucose