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Phosphorylation of glycolytic and gluconeogenic enzymes by the insulin receptor kinase
Abstract:
Various glycolytic and gluconeogenic enzymes were tested as substrates for the insulin receptor kinase. Phosphofructokinase and phosphoglycerate mutase were found to be the best substrates. Phosphorylation of these enzymes was rapid, stimulated 2- to 6-fold by 10(-7) M insulin and occurred exclusively on tyrosine residues. Enolase, fructose 1,6-bisphosphatase, lactate dehydrogenases in decreasing order, were also subject to insulin-stimulated phosphorylation but to a smaller extent than that for phosphofructokinase or phosphoglycerate mutase. The phosphorylation of phosphofructokinase was studied most extensively since phosphofructokinase is known to catalyze a rate-limiting step in glycolysis. The apparent Km of the insulin receptor for phosphofructokinase was 0.1 microM, which is within the physiologic range of concentration of this enzyme in most cells. Tyrosine phosphorylation of phosphofructokinase paralleled autophosphorylation of the beta-subunit of the insulin receptor with respect to time course, insulin dose response (half maximal effect between 10(-9) and 10(-8) M insulin), and cation requirement (Mn2+ greater than Mg2+ much greater than Ca2+). Further study will be required to determine whether the tyrosine phosphorylation of phosphofructokinase plays a role in insulin-stimulated increases in glycolytic flux.
Insights
Insulin receptor kinase phosphorylates key glycolytic enzymes, notably phosphofructokinase, on tyrosine residues. This phosphorylation, stimulated by insulin, may influence glycolysis regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Metabolism
Background:
- Insulin signaling regulates glucose metabolism through complex pathways.
- The insulin receptor kinase plays a crucial role in mediating insulin's effects.
- Identifying direct enzyme substrates of the insulin receptor kinase is key to understanding metabolic regulation.
Purpose of the Study:
- To investigate which glycolytic and gluconeogenic enzymes serve as substrates for the insulin receptor kinase.
- To characterize the insulin-stimulated phosphorylation of these enzymes, particularly phosphofructokinase.
Main Methods:
- Enzymatic assays using various glycolytic and gluconeogenic enzymes as substrates for purified insulin receptor kinase.
- Analysis of phosphorylation sites (tyrosine residues) and kinetic parameters (apparent Km).
- Comparison of enzyme phosphorylation with insulin receptor beta-subunit autophosphorylation.
Main Results:
- Phosphofructokinase and phosphoglycerate mutase were identified as the best substrates for insulin receptor kinase.
- Insulin (10(-7) M) stimulated the rapid tyrosine phosphorylation of these enzymes 2- to 6-fold.
- Phosphorylation kinetics and insulin dose-response for phosphofructokinase mirrored insulin receptor autophosphorylation.
Conclusions:
- The insulin receptor kinase directly phosphorylates key glycolytic enzymes, including phosphofructokinase.
- This tyrosine phosphorylation is insulin-dependent and occurs at physiologically relevant enzyme concentrations.
- The functional significance of phosphofructokinase tyrosine phosphorylation in insulin-mediated glycolysis requires further investigation.