N-Terminal Mutations Modulate Yeast SNF1 Protein Kinase Function

INVESTIGATIONS

N-Terminal Mutations Modulate Yeast SNF1 Protein Kinase Function

F. Estruch, M. A. Treitel, X. Yang and M. Carlson
Department of Genetics and Development and Institute of Cancer Research, Columbia University, College of Physicians and Surgeons, New York, New York 10032

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 proteins 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.

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				N. Nath, R. R. McCartney, and M. C. Schmidt Yeast Pak1 Kinase Associates with and Activates Snf1 Mol. Cell. Biol., June 1, 2003; 23(11): 3909 - 3917. [Abstract] [Full Text] [PDF]

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				K. Ludin, R. Jiang, and M. Carlson Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae PNAS, May 26, 1998; 95(11): 6245 - 6250. [Abstract] [Full Text] [PDF]

				R Jiang and M Carlson Glucose regulates protein interactions within the yeast SNF1 protein kinase complex. Genes & Dev., December 15, 1996; 10(24): 3105 - 3115. [Abstract] [PDF]

				S. A. Hawley, M. Davison, A. Woods, S. P. Davies, R. K. Beri, D. Carling, and D. G. Hardie Characterization of the AMP-activated Protein Kinase Kinase from Rat Liver and Identification of Threonine 172as the Major Site at Which It Phosphorylates AMP-activated Protein Kinase J. Biol. Chem., November 1, 1996; 271(44): 27879 - 27887. [Abstract] [Full Text] [PDF]

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