N-terminal mutations modulate yeast SNF1 protein kinase function.
The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-delta N kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-delta N protein showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell.
Estruch
F
F
Department of Genetics and Development, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
Treitel
M A
MA
Yang
X
X
Carlson
M
M
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins; D002850:Chromatography, Gel; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005799:Genes, Dominant; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism
D002352:Carrier Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; C078651:SNF4 protein, S cerevisiae; D055372:AMP-Activated Protein Kinases