Protein dephosphorylation by phosphatase PP1 plays a central role in mediating the effects of insulin on glucose and lipid metabolism. A PP1C-targeting protein expressed in 3T3-L1 adipocytes (called PTG, for protein targeting to glycogen) was cloned and characterized. PTG was expressed predominantly in insulin-sensitive tissues. In addition to binding and localizing PP1C to glycogen, PTG formed complexes with phosphorylase kinase, phosphorylase a, and glycogen synthase, the primary enzymes involved in the hormonal regulation of glycogen metabolism. Overexpression of PTG markedly increased basal and insulin-stimulated glycogen synthesis in Chinese hamster ovary cells overexpressing the insulin receptor, which do not express endogenous PTG. These results suggest that PTG is critical for glycogen metabolism, possibly functioning as a molecular scaffold.

No abstract

Spinal muscular atrophy (SMA) is caused by deletion or mutation of both copies of the SMN1 gene which produces an essential protein known as SMN. The severity of SMA is modified by variable copy number of a second gene, SMN2 that produces an mRNA that is incorrectly spliced with deletion of the last exon. We described previously the discovery of potent C5-substituted quinazolines that increase SMN2 gene expression by two-fold. Discovery of potent SMN2 promoter inducers relied on a cellular assay without knowledge of the molecular target. Using protein microarray scanning with a radiolabeled C5-quinazoline probe, we identified the scavenger decapping enzyme, DcpS as a potential binder. We show that the C5-quinazolines potently inhibit DcpS decapping activity, and that the potency of inhibition correlates with potency for SMN2 promoter induction. Binding of C5-quinazolines to DcpS holds the enzyme in an open, catalytically incompetent conformation. DcpS is a nuclear shuttling protein that binds and hydrolyzes the m7GpppN mRNA cap structure and a modulator of RNA metabolism. Therefore DcpS represents a novel therapeutic target for modulating gene expression by a small molecule.

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...

The human pregnane X nuclear receptor (PXR) is a xenobioticregulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and

Utilizing a genetic screen in the yeast Saccharomyces cerevisiae, we identified a novel autoactivation region in mammalian MEK1 that is involved in binding the specific MEK inhibitor, PD 184352. The genetic screen is possible due to the homology between components of the yeast pheromone response pathway and the eukaryotic Raf-MEK-ERK signaling cascade. Using the FUS1::HIS3 reporter as a functional readout for activation of a reconstituted Raf-MEK-ERK signaling cascade, randomly mutagenized MEK variants that were insensitive to PD 184352 were obtained. Seven single-base-change mutations were identified, five of which mapped to kinase subdomains III and IV of MEK. Of the seven variants, only one, a leucine-to-proline substitution at amino acid 115 (Leu115Pro), was completely insensitive to PD 184352 in vitro (50% inhibitory concentration >10 M). However, all seven mutants displayed strikingly high basal activity compared to wild-type MEK. Overexpression of the MEK variants in HEK293T cells resulted in an increase in mitogen-activated protein (MAP) kinase phosphorylation, a finding consistent with the elevated basal activity of these constructs. Further, treatment with PD 184352 failed to inhibit Leu115Pro-stimulated MAP kinase activation in HEK293T cells, whereas all other variants had some reduction in phospho-MAP kinase levels. By using cyclic AMP-dependent protein kinase (1CDK) as a template, an MEK homology model was generated, with five of the seven identified residues clustered together, forming a potential hydrophobic binding pocket for PD 184352. Additionally, the model allowed identification of other potential residues that would interact with the inhibitor. Directed mutation of these residues supported this region's involvement with inhibitor binding.The mitogen-activated protein (MAP) kinase cascade, comprised of MAP kinase (ERK), MAP kinase kinase (MEK), and MAP kinase kinase kinase (Raf), is an evolutionarily conserved signaling module that regulates growth, differentiation, and movement in eukaryotic cells in response to extracellular stimulation (reviewed in reference 23). A key regulatory component of this pathway is MAP kinase kinase, or MEK, a dualspecificity kinase that phosphorylates MAP kinase (ERK) on specific threonine and tyrosine residues. This phosphorylation activates MAP kinase and induces a host of downstream cellular responses (reviewed in reference 10).MEK itself, is subject to regulation and activation by Raf phosphorylation on two serine residues, Ser218 and Ser222 (2, 33), which lie in a regulatory loop between conserved kinase subdomains VII and VIII (16). Substitution of these serine residues with negatively charged amino acids, such as aspartate or glutamate, partially mimics the phosphorylation modification and results in a constitutively active kinase, presumably through stabilization of the regulatory loop, allowing the enzyme to retain an active conformation (2).Another regulatory feature of MEK is a proline-rich region, located carboxy terminal to the regulatory loop, whic...

Protein targeting to glycogen (PTG) is a scaffolding protein that targets protein phosphatase 1α (PP1α) to glycogen, and links it to enzymes involved in glycogen synthesis and degradation. We generated mice that possess a heterozygous deletion of the PTG gene. These mice have reduced glycogen stores in adipose tissue, liver, heart, and skeletal muscle, corresponding with decreased glycogen synthase activity and glycogen synthesis rate. Although young PTG heterozygous mice initially demonstrate normal glucose tolerance, progressive glucose intolerance, hyperinsulinemia, and insulin resistance develop with aging. Insulin resistance in older PTG heterozygous mice correlates with a significant increase in muscle triglyceride content, with a corresponding attenuation of insulin receptor signaling. These data suggest that PTG plays a critical role in glycogen synthesis and is necessary to maintain the appropriate metabolic balance for the partitioning of fuel substrates between glycogen and lipid

Ste7p and Mkk1p are MEK (MAPK/ERK kinase) family members that function in the mating and cell integrity signal transduction pathways in Saccharomyces cerevisiae. We selected STE7 and MKK1 mutations that stimulated their respective pathways in the absence of an inductive signal. Strikingly, serine-to-proline substitutions at analogous positions in Ste7p (position 368) and Mkk1p (position 386) were recovered by independent genetic screens. Such an outcome suggests that this substitution in other MEKs would exhibit similar properties. The Ste7p-P 368 variant has higher basal enzymatic activity than Ste7p but still requires induction to reach full activation. The higher activity associated with Ste7p-P 368 allows it to compensate for defects in the cell integrity pathway, but it does so only when it is overproduced or when Ste5p is missing. This behavior suggests that Ste5p, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7p specificity.