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.
Mesh Terms:
AMP-Activated Protein Kinases, Amino Acid Sequence, Base Sequence, Carrier Proteins, Chromatography, Gel, Enzyme Activation, Fungal Proteins, Genes, Dominant, Immunologic Techniques, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphorylation, Protein Kinases, Protein Processing, Post-Translational, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors
AMP-Activated Protein Kinases, Amino Acid Sequence, Base Sequence, Carrier Proteins, Chromatography, Gel, Enzyme Activation, Fungal Proteins, Genes, Dominant, Immunologic Techniques, Molecular Sequence Data, Mutagenesis, Site-Directed, Phosphorylation, Protein Kinases, Protein Processing, Post-Translational, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors
Genetics
Date: Nov. 01, 1992
PubMed ID: 1468623
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