Reversible protein phosphorylation is one of the most pervasive strategies for regulation in biological systems. An example of this form of regulation is provided by the regulatory phosphorylation of isocitrate dehydrogenase (IDH) of Escherichia coli. This has been chosen as the experimental model for this study primarily because of the potential for the use of molecular genetics and recombinant DNA technology in probing the system. The ultimate goal of this project is to achieve an understanding of the functioning of the phosphorylation system in the intact organism. This will be accomplished by characterizing the behavior of the individual components and using this knowledge to analyze the functioning of the system in vivo. Studies performed under this proposal will concern the structure/function relationships of IDH kinase and phosphatase and the bahavior of these activities in vivo. IDH kinase and phosphatase have been found to be physically associated, probably on the same polypeptide chain. In an effort to determine the regulatory and structural significance of this bifunctionality, the relationships between the functional domains will be characterized. In addition to the methods of protein chemistry, this study will make extensive use of recombinant DNA technology, including in vitro mutagenesis. Specific problems to be addressed include the evolutionary relationship between the domains, the degree to which they are physically autonomous and the potential for inter-domain allosteric communication. The clones of the IDH kinase/phosphatase gene and of its genetically altered derivatives which will be developed and characterized in the structural studies, will provide the basis for study of the system in vivo. In this project, variation in the cellular level of IDH kinase/phosphatase will be used to define the factors which are responsible for the rate at which the phosphorylation system responds to stimulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033927-03
Application #
3284117
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1984-07-01
Project End
1987-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Chung, T; Resnik, E; Stueland, C et al. (1993) Relative expression of the products of glyoxylate bypass operon: contributions of transcription and translation. J Bacteriol 175:4572-5
Ikeda, T P; Houtz, E; LaPorte, D C (1992) Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity. J Bacteriol 174:1414-6
Ikeda, T; LaPorte, D C (1991) Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity. J Bacteriol 173:1801-6
Resnik, E; LaPorte, D C (1991) Introduction of single-copy sequences into the chromosome of Escherichia coli: application to gene and operon fusions. Gene 107:19-25
Laporte, D C; Stueland, C S; Ikeda, T P (1989) Isocitrate dehydrogenase kinase/phosphatase. Biochimie 71:1051-7
Stueland, C S; Ikeda, T P; LaPorte, D C (1989) Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase. J Biol Chem 264:13775-9
Klumpp, D J; Plank, D W; Bowdin, L J et al. (1988) Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase. J Bacteriol 170:2763-9
Chung, T; Klumpp, D J; LaPorte, D C (1988) Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map. J Bacteriol 170:386-92
Stueland, C S; Gorden, K; LaPorte, D C (1988) The isocitrate dehydrogenase phosphorylation cycle. Identification of the primary rate-limiting step. J Biol Chem 263:19475-9
Stueland, C S; Eck, K R; Stieglbauer, K T et al. (1987) Isocitrate dehydrogenase kinase/phosphatase exhibits an intrinsic adenosine triphosphatase activity. J Biol Chem 262:16095-9