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Organizing glucose disposal: emerging roles of the glycogen targeting subunits of protein phosphatase-1.

Glucose is stored in mammalian tissues in the form of glycogen. Glycogen levels are markedly reduced in liver or muscle cells of patients with insulin-resistant or insulin-deficient forms of diabetes, suggesting that impaired glycogen synthesis may contribute to development of hyperglycemia. Recently, interest in this area has been further stimulated by new insights into the spatial organization of metabolic enzymes within cells and the importance of such organization in regulation of glycogen metabolism. It is now clear that a four-member family of glycogen targeting subunits of protein phosphatase-1 (PP1) plays a major role in coordinating these events. These proteins target PP1 to the glycogen particle and also bind differentially to glycogen synthase, glycogen phosphorylase, and phosphorylase kinase, thereby serving as molecular scaffolds. Moreover, the various glycogen-targeting subunits have distinct tissue expression patterns and can influence regulation of glycogen metabolism in response to glycogenic and glycogenolytic signals. The purpose of this article is to summarize new insights into the structure, function, regulation, and metabolic effects of the glycogen-targeting subunits of PP1 and to evaluate the possibility that these molecules could serve as therapeutic targets for lowering of blood glucose in diabetes.

Animals↗

Impaired surfactant synthesis in fetal type II lung cells from gsd/gsd rats.

The importance of intracellular glycogen for surfactant synthesis was investigated in fetal type II lung cells isolated from rats with a glycogen storage disorder, designated gsd/gsd. Compared to cells from a control Wistar strain, cultured gsd/gsd pneumocytes were glycogenrich and contained fewer and smaller lamellar inclusions. Freshly isolated cells from day 19-21 fetuses of control rats demonstrated the expected gestational rise and fall of cellular glycogen seen in intact fetal lungs. At day 20, when tissue glycogen peaks, cellular glycogen content was 48 and 70 nmol glucose/micrograms DNA in isolated type II cells of control and gsd/gsd lungs, respectively. In control cells, while active glycogen phosphorylase changed from 35 to 65% of total during 24 h of culture, glycogen fell 85%. In contrast, gsd/gsd cell phosphorylase was not activated, phosphorylase kinase activity was nondetectable, and glycogen per cell remained unchanged. [3H]Choline incorporation into total PC and disaturated PC (DSPC) was 50 and 62% lower, respectively, in gsd/gsd type II cells compared to controls in the absence of exogenous substrate. Cellular content of the surfactant-associated protein SP-A was similar in control and gsd/gsd cells at day 20, and increased 3- to 4-fold during a subsequent 24-h interval of tissue culture. These results suggest that PC synthesis is dramatically impaired in type II cells in which glycogen cannot be mobilized, but SP-A is synthesized at normal rates. This work characterizes the isolated gsd/gsd fetal type II cell and supports its use in future studies to determine the importance and relative utilization of specific nonglycogen substrates.

Animals↗

Purification and characterization of smooth muscle myosin-associated phosphatase from chicken gizzards.

Myosin light chain phosphatase associated with smooth muscle myosin (MAPP) was isolated from chicken gizzard. The MAPP was tightly associated with myosin and was not dissociated from myosin under the physiological ionic conditions. The phosphatase was dissociated from myosin in the presence of high MgCl2, i.e. 80 mM MgCl2. The binding site of the enzyme on the myosin molecule was the subfragment-2 region, since the enzyme did bind to the myosin rod and heavy meromyosin but not to the subfragment-1 affinity column. MAPP was purified with a heparin-Sepharose 6B column, and two activity peaks were obtained, i.e. MAPP I and MAPP II. The major activity peak, MAPP I, was further purified to homogeneity by thiophosphorylated myosin light chain-Sepharose 4B column chromatography. MAPP I was a tetramer composed of four 34-kDa subunits. The enzyme preferentially dephosphorylated the beta-subunit of phosphorylase kinase and was strongly inhibited by the heat- and acid-stable protein phosphatase inhibitor-1, whereas it was partially inhibited by the inhibitor-2. The IC50 (concentration of inhibitor giving 50% inhibition) value for the inhibition of the enzyme by okadaic acid was 70 nM which was about eight times higher than skeletal muscle type-1 and 390 times higher than type-2 protein phosphatase. These results demonstrate that the MAPP I is a type-1-like protein phosphatase, although the properties are not the same as type-I phosphatase. The properties of the myosin-associated phosphatase were distinct from the phosphatases reported previously, although some properties were similar to smooth muscle phosphatase-IV. Therefore, it is concluded that MAPP I is a novel smooth muscle protein phosphatase. Since it strongly associated with smooth muscle myosin, it is likely that MAPP I is responsible for the dephosphorylation of smooth muscle myosin in situ.

Adenosine Triphosphate↗

Turkey gizzard smooth muscle myosin phosphatase-III is a novel protein phosphatase.

Chromatography of turkey gizzard extract on Sephacryl S-300 has been shown to fractionate the various smooth muscle phosphatases. We have previously reported the purification and characterization of three of these enzymes, termed smooth muscle phosphatase (SMP)-I, -II, and -IV. Recently, we have purified SMP-III to near homogeneity. Although all of the smooth muscle phosphatases dephosphorylate the isolated myosin light chains, only SMP-III and -IV are active toward intact myosin and, therefore, are most likely to play a direct role in the muscle contraction-relaxation process. SMP-III has a higher molecular weight (390,000), as determined by gel filtration, than the other smooth muscle phosphatases and migrates as single band with a molecular weight of 40,000 in a sodium dodecyl sulfate-polyacrylamide gel. SMP-III is immunologically distinct from SMP-I and -II. It dephosphorylates heavy meromyosin and the isolated myosin light chains at a rapid rate but has low activity toward phosphorylase alpha. The activity of SMP-III is not affected by Ca2+ but is activated by Mn2+.Mg2+ stimulates the activity toward heavy meromyosin but inhibits the myosin light chain phosphatase activity. Attempts to classify SMP-III according to the scheme proposed by Ingebritsen and Cohen (Ingebritsen T. S., and Cohen, P. (1983) Science 221, 331-338) revealed that it is resistant to the heat stable inhibitor-2, suggesting that it is a Type 2 protein phosphatase. However, SMP-III is inhibited by concentrations of okadaic acid which are characteristic of Type 1 protein phosphatases and it binds to heparin-Sepharose like other Type 1 phosphatases. But most interestingly, SMP-III does not dephosphorylate the alpha- or beta-subunits of phosphorylase kinase, a property not reported for any Ser/Thr protein phosphatase.

Animals↗

Identification, purification, and characterization of a novel serine/threonine protein phosphatase from bovine brain.

A novel serine/threonine protein phosphatase is identified, and the catalytic subunit, obtained from a detergent extraction of the pellet generated by a 100,000 x g centrifugation of a whole bovine brain homogenate, is purified and characterized. The protein phosphatase, designated as PP3, has a Mr of 36,000, does not require divalent cations for activity, is stimulated rather than inhibited by inhibitor 2, is inhibited by both okadaic acid and microcystin-LR with an intermediate IC50 compared to type 1 and type 2A protein phosphatases, and preferentially dephosphorylates the beta subunit of phosphorylase kinase. Substrate specificity, immunoblotting with type-specific antisera, and the amino acid sequences of peptides derived from PP3 indicate that PP3 is not an isoform of any known serine/threonine protein phosphatase.

Amino Acid Sequence↗

[A case of glycogen storage myopathy with acute heart failure].

A case of 25-year-old woman with glycogen storage myopathy is reported here. She was hospitalized for acute heart failure after alcohol drinking. The electrocardiogram on admission showed marked ST elevation. Laboratory data showed elevated levels of serum myogenic enzymes but no rise in cardiomyogenic enzyme: CK 3862 IU/l CK-MB 35 IU/l, LDH 427 IU/l, GOT 203 IU/l. After several days, she recovered from acute heart failure and could walk without supporting. ST elevation in ECG and elevated myogenic enzymes were also normalized. The occurrence of acute myocardial infarction was ruled out because a coronary angiogram and 99 Tcm scintigram were normal. Physical examination revealed proximal muscular weakness and mental retardation (WAIS, total 72). Venous lactate response was normal after semi-ischemic forearm exercise. PAS staining of muscle specimen showed an excess deposit of glycogen. Ragged-red fibers were not seen on Gomori-trichrome stain. By electron microscopy, a large amount of glycogen particles were demonstrated in the subsarcolemma, but there were no abnormal mitochondrial changes. Biochemical analysis showed accumulation of glycogen in muscles: 28.7 mg/g muscle (normal 11.4 +/- 4.2 mg/g muscle). The activities of enzyme in the pathway of glycogen and glycogenosis (alpha-glucosidase, amylo-1,6-glucosidase, phosphorylase a, phosphorylase kinase, phosphofructokinase, etc.) were within normal limits. The spectrum of glycogen iodine complex was normal. Our case was different from any type of muscle glycogen storage disease previously reported. The etiology of an excess of glycogen deposit in muscles is unknown.

Acute Disease↗

Identification of protein phosphatases 1 and 2B as ribosomal protein S6 phosphatases in vitro and in vivo.

Protein phosphatases 1 and 2B from rabbit skeletal muscle were found to catalyze the dephosphorylation of ribosomal protein S6 in vitro. Phosphorylation of protein phosphatase-1 by the transforming protein of Rous sarcoma virus, pp60v-src, abolished S6 dephosphorylation by the purified enzyme. Analysis of the dephosphorylation of phosphorylase a and phosphorylase kinase in Xenopus oocyte extracts and after microinjection indicated the presence of oocyte enzymes similar to protein phosphatases-1 and -2B. Studies with 32P-labeled 40 S ribosomal subunits suggested that these enzymes were functioning as S6 phosphatases in oocytes. These findings support the hypothesis that regulation of protein phosphatase activity may be involved in the increase in S6 phosphorylation observed after mitogenic stimulation.

Animals↗

[Changes in glycogen metabolism in hereditary muscular diseases (review)].

Impairments of glycogen metabolism in muscles, which occurred in some types of the glycogen storage disease as well as in hereditary diseases of unestablished biochemical nature are reviewed. Hereditary diseases of animals caused by impairment of glycogen metabolism are considered.

Adult↗

Demonstration of different metal ion-induced calcineurin conformations using a monoclonal antibody.

It has been suggested that calcineurin, a calmodulin-stimulated phosphatase, may exist in different metal ion-dependent conformational states (Pallen, C.J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141). Evidence in favor of this hypothesis comes from studies involving a monoclonal antibody, VA1, which is specific for the small (beta) subunit of calcineurin. This antibody inhibits Ni2+-stimulated but not Mn2+-stimulated phosphatase activity against p-nitrophenyl phosphate and phosphorylase kinase. Inhibition is not due to competition of the antibody with substrate or to interference with metal ion binding to the enzyme. Complex formation between the antibody and calcineurin can be demonstrated either in the presence of Mn2+ or Ni2+ or in the absence of metal ion activators. These results indicate that the active conformational states of calcineurin are metal ion dependent, that the monoclonal antibody VA1 affects the Ni2+-induced conformational change of the enzyme, and that the beta subunit of calcineurin plays a critical role in the expression of Ni2+-stimulated phosphatase activity.

Animals↗

Thermodynamics of the binding of AMP to glycogen phosphorylase a.

The binding of AMP to rabbit muscle glycogen phosphorylase a (EC 2.4.1.1.) has been studied by equilibrium dialysis and isothermal microcalorimetry at pH 6.9 over a temperature range of 25 degrees C to 35 degrees C. Thermal titration experiments were carried out in various buffer systems. We have found by these methods that a certain number of protons are released when the protein binds to the ligand and are taken up by the buffer. The tetramer of phosphorylase a has been shown to have four equal and independent, non-cooperative binding sites for AMP at 25 degrees C, 30 degrees C, and 35 degrees C; these sites can be assigned to the so-called nucleotide or, activator, sites in the protein. The binding constants together with the changes in Gibbs energy, enthalpy, and entropy per site for the AMP binding were calculated at each temperature. A negative delta Cp value of -2.3 +/- 0.2 J K-1 (AMP bound)-1 was obtained for this binding process. The hydrophobic and vibrational contributions of the heat capacity and entropy changes have been resolved by the method described by Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). From this analysis, it appears that the binding is, in all cases, enthalpy-driven, the two entropic contributions, hydrophobic and vibrational, having opposing effects.

Adenosine Monophosphate↗

Acute uremia following dietary potassium depletion. II. Effect on tissue carbohydrate composition.

In order to evaluate the potential role of hyperkalemia and metabolic acidosis on the disturbances of carbohydrate metabolism normally seen in uremia, a specific model of acute uremia devoid of hyperkalemia and severe metabolic acidosis was chosen. Therefore, rats were deprived of potassium prior to induction of acute uremia. Potassium depletion caused a significant decrease of muscle and liver glycogen due to activation of phosphorylase kinase, whereas glycogen concentration in heart muscle was unchanged and elevated in the kidney of sham-operated and ureter-ligated animals. In contrast, glucose concentrations were enhanced in the liver and the kidney, unchanged in heart muscle and decreased in skeletal muscle. We conclude that carbohydrate abnormalities occur in acute uremia despite normokalemia and mild metabolic acidosis. Furthermore, acute uremia accompanied by prior potassium depletion results in no net effect on cardiac glycogen metabolism but stimulates glycogenolysis in both skeletal muscle and the liver.

Animals↗