Regulatory domains of Snf1-activating kinases determine pathway specificity.
In Saccharomyces cerevisiae, the Snf1 kinase can be activated by any one of three upstream kinases, Sak1, Tos3, or Elm1. All three Snf1-activating kinases contain serine/threonine kinase domains near their N termini and large C-terminal domains with little sequence conservation and previously unknown function. Deletion of the C-terminal domains of Sak1 and Tos3 greatly reduces their ability to activate the Snf1 pathway. In contrast, deletion of the Elm1 C-terminal domain has no effect on Snf1 signaling but abrogates the ability of Elm1 to participate in the morphogenetic-checkpoint signaling pathway. Thus, the C-terminal domains of Sak1, Tos3, and Elm1 help to determine pathway specificity. Additional deletion mutants of the Sak1 kinase revealed that the N terminus of the protein is essential for Snf1 signaling. The deletion of 43 amino acids from within the N terminus of Sak1 (residues 87 to 129) completely blocks Snf1 signaling and activation loop phosphorylation in vivo. The Sak1 kinase domain (lacking both N-terminal and C-terminal domains) is catalytically active and specific in vitro but is unable to promote Snf1 signaling in vivo when expressed at normal levels. Our studies indicate that the kinase domains of the Snf1-activating kinases are not sufficient by themselves for their proper function and that the nonconserved N-terminal and C-terminal domains are critical for the biological activities of these kinases.
Rubenstein
Eric M
EM
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
McCartney
Rhonda R
RR
Schmidt
Martin C
MC
D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*
D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C071570:SNF1-related protein kinases; C094326:PRK1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C502450:TOS3 protein, S cerevisiae