Generation of a dominant-negative glycogen targeting subunit for protein phosphatase-1.
Modulation of the expression of the protein phosphatase-1 (PP1) glycogen-targeting subunit PTG exerts profound effects on cellular glycogen metabolism in vitro and in vivo. PTG contains three distinct binding domains for glycogen, PP1, and a common site for glycogen synthase and phosphorylase. The impact of disrupting the PP1-binding domain on PTG function was examined in 3T3-L1 adipocytes. A full-length PTG mutant was generated as an adenoviral construct in which the valine and phenylalanine residues in the conserved PP1-binding domain were mutated to alanine (PTG-VF). Infection of fully differentiated 3T3-L1 adipocytes with the PTG-VF adenovirus reduced glycogen stores by over 50%. In vitro, PTG-VF competitively interfered with wild-type PTG action, suggesting that the mutant construct acted as a dominant-negative molecule. The reduction in cellular glycogen storage was due to a significantly increased rate of glycogen turnover. Interestingly, acute basal and insulin-stimulated glucose uptake and glycogen synthesis rates were enhanced in PTG-VF expressing cells vs. control 3T3-L1 adipocytes, likely as a compensatory response to the loss of glycogen stores. These results indicate that the mutation of the PP1-binding domain on PTG resulted in the generation of a dominant-negative molecule that impeded endogenous PTG action and reduced cellular glycogen levels, through enhancement of glycogenolysis rather than impairment of glycogen synthesis.
Jurczak
Michael J
MJ
Department of Medicine, Section of Endocrinology, Diabetes, and Metabolism and the Committee on Molecular Metabolism and Nutrition, The University of Chicago, Chicago, Illinois, USA.
Zapater
Joseph L
JL
Greenberg
Cynthia C
CC
Brady
Matthew J
MJ
D041721:3T3-L1 Cells; D000256:Adenoviridae; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism*; D006003:Glycogen / Q000378:metabolism*; D006006:Glycogen Synthase / Q000378:metabolism*; D050261:Glycogenolysis / Q000235:genetics* / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D054645:Protein Phosphatase 1 / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; D006003:Glycogen; D006006:Glycogen Synthase; D054645:Protein Phosphatase 1; D005947:Glucose