Identification of binding sites on protein targeting to glycogen for enzymes of glycogen metabolism

The activation of protein phosphastase-1 (PP1) by insulin-plays a critical role in the regulation of glycogen metabolism. PTG is a PP1 glycogen-target ing protein, which also binds the PP1 substrates glycogen synthase, glycoge n phosphorylase, and phosphorylase kinase (Printen, J.:A,, Brady, M. J., an d Saltiel, A. R. (1997) Science 275, 1475-1478). Through a combination of d eletion analysis and site-directed mutagenesis, the regions on PTG responsi ble for binding PP1 and its substrates have been delineated. Mutagenesis of Val-62 and Phe-64 in the highly conserved (K/R)VXF PP1-binding motif to al anine was sufficient to ablate PP1 binding to PTG. Phosphorylase kinase, gl ycogen synthase, and phosphorylase binding all mapped to the same C-termina l region of PTG. Mutagenesis of Asp-225 and Glu-228 to alanine completely b locked the interaction between PTG and these three enzymes, without affecti ng PP1 binding. Disruption of either PP1 or substrate binding to PTG blocke d the stimulation of PP1 activity in vitro against phosphorylase, indicatin g that both binding sites may be important in PTG action. Transient overexp ression of wild type PTG in Chinese hamster ovary cells overexpressing the insulin receptor caused a 50-fold increase in glycogen levels. Expression o f PTG mutants that do not bind PP1 had no effect on glycogen accumulation, indicating that PP1 targeting is essential for PTG function. Likewise, expr ession of the PTG mutants that do not bind PP1 substrates did not increase glycogen levels, indicating that PP1 targeting glycogen is not sufficient f or the metabolic effects of PTG, These results cumulatively demonstrate tha t PTG serves as a molecular scaffold, allowing PP1 to recognize its substra tes at the glycogen particle.