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Generation of a Dominant‐Negative Glycogen Targeting Subunit for Protein Phosphatase‐1
Author(s) -
Jurczak Michael J.,
Zapater Joseph L.,
Greenberg Cynthia C.,
Brady Matthew J.
Publication year - 2010
Publication title -
obesity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.438
H-Index - 199
eISSN - 1930-739X
pISSN - 1930-7381
DOI - 10.1038/oby.2010.32
Subject(s) - glycogen , glycogen synthase , glycogen phosphorylase , glycogenolysis , glycogen debranching enzyme , protein phosphatase 1 , glycogen branching enzyme , biology , protein subunit , mutant , chemistry , phosphatase , endocrinology , medicine , biochemistry , phosphorylation , gene
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.