Role of Protein Targeting to Glycogen (PTG) in the Regulation of Protein Phosphatase-1 Activity

Abstract

We have recently cloned from 3T3-L1 adipocytes a novel glycogen-targeting subunit of protein phosphatase-1, termed PTG (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes resulted in a marked increase in PTG expression. Immobilized glutathione S-transferase (GST)-PTG fusion protein specifically bound either PP1 or phosphorylase a. Addition of soluble GST-PTG to 3T3-L1 lysates increased PP1 activity against 32 P-labeled phosphorylase a by decreasing the K m of PP1 for phosphorylase 5-fold, while having no effect on the V max of the dephosphorylation reaction. Alternatively, PTG did not affect PP1 activity against hormone-sensitive lipase. PTG was not a direct target of intracellular signaling, as insulin or forskolin treatment of cells did not activate a kinase capable of phosphorylating PTG in vivo or in vitro. Finally, PTG decreased the ability of DARPP-32 to inhibit PP1 activity from 3T3-L1 adipocyte lysates. These data cumulatively suggest that PTG increases PP1 activity against specific proteins by several distinct mechanisms.While much attention has been focused on the activation of protein kinase signaling cascades, many enzymes involved in glucose and lipid metabolism are regulated by dephosphorylation (2). As the main physiological hormone controlling glucose utilization, insulin exerts many of its effects through the activation of type 1 protein phosphatase (PP1).1 However, insulin treatment of cells results in the dephosphorylation of only a limited number of proteins, while simultaneously other proteins are phosphorylated (3). This paradox suggests that mechanisms must exist whereby insulin activates discrete pools of PP1, leading to the dephosphorylation of specific target proteins.PP1 is found in many cellular compartments, including the nucleus, plasma membrane, and glycogen particle. It is thought that the cellular localization of this enzyme is mediated by its association with targeting proteins (4, 5). We have recently identified a novel PP1 glycogen-targeting subunit from 3T3-L1 adipocytes, termed PTG for protein targeting to glycogen (1). PTG is the third member of a family of PP1 glycogen-targeting subunits, which also includes R GL , isolated from muscle (6, 7), and the hepatic G L protein (8, 9). In contrast to the restricted localization of R GL and G L , PTG is highly expressed in all insulin-sensitive tissues.2 In addition to targeting PP1 to the glycogen particle, PTG can also form complexes with PP1 substrate enzymes that regulate glycogen metabolism, namely glycogen synthase, glycogen phosphorylase, and phosphorylase kinase. Overexpression of PTG in the metabolically inactive CHO-IR cell line dramatically increased the levels of basal and insulin-stimulated glycogen synthesis (1). PTG may therefore serve as a scaffolding protein, assembling the proteins involved in glycogen metabolism and priming them for the reception of intracellular signals.The mechanism by which insulin sp...

Biophysical interaction between phospholamban and protein phosphatase 1 regulatory subunit GM

“…In addition to targeting PP1 to the glycogen particle, PTG can also form complexes with PP1 substrate enzymes that regulate glycogen metabolism, namely glycogen synthase, glycogen phosphorylase, and phosphorylase kinase. In this case, PTG not only binds PP1C and glycogen but also co-localises PP1 with its substrate at the glycogen particles suggesting that PTG is responsible for assembling metabolic enzymes for the localised reception of intracellular signals [23,24]. In a similar way, the present experiments suggest that GM, which targets PP1C to both glycogen particles and the SR of striated muscle [13], may localise PP1C close to its PLB substrate in order to control speci¢cally the activation of the calcium ATPase.…”

Disruption of the Striated Muscle Glycogen Targeting Subunit PPP1R3A of Protein Phosphatase 1 Leads to Increased Weight Gain, Fat Deposition, and Development of Insulin Resistance

“…The expression of hepatic R5/PTG is downregulated in streptozotocininduced diabetes and restored by insulin treatment (20), but muscle R5/PTG is not altered by these treatments (21). In addition, R5/PTG is not known to be acutely regulated by insulin or epinephrine (22). R6 (33 kDa, the product of the PPP1R3D gene) is present in a wide variety of tissues, and its expression was not altered in streptozotocininduced diabetes (18,20).…”

Fatty Acid Infusion Selectively Impairs Insulin Action on Akt1 and Protein Kinase C λ/ζ but Not on Glycogen Synthase Kinase-3

“…The reaction was initiated by adding 15 g of 32 P-labeled phosphorylase a in the presence of 3 nM okadaic acid and 5 mM caffeine. The phosphate release was determined as described previously (52).…”