Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes.

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise.

In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction, in vivo treadmill running and in situ electrical stimulation. Both procedures resulted in a 2-fold increase in the GS -/+ glucose-6-P activity ratio in WT mice, but this response was completely absent in the KO mice. The KO mice, which also have a reduced GS activity associated with significantly reduced basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R(GL) OE mice are characterized by enhanced GS activity ratio and an approximately 3-4-fold increase in glycogen content in skeletal muscle. These animals were able to tolerate exercise normally. Stimulation of GS and glucose uptake following muscle contraction was not significantly different as compared with WT littermates. These results indicate that although PP1G/R(GL) is not necessary for activation of GS by insulin, it is essential for regulation of glycogen metabolism under basal conditions and in response to contractile activity, and may explain the reduced muscle glycogen content in the R(GL) KO mice, despite the normal insulin activation of GS.

Gene structure and expression of the targeting subunit, RGL, of the muscle-specific glycogen-associated type 1 protein phosphatase, PP1G.

The type I phosphatase associated with glycogen, PP1G, plays an important role in glycogen metabolism. PP1G is targeted to glycogen by the R(GL) subunit, which regulates the function of the enzyme. We report the cloning and characterization of the gene as well as the pattern of expression of the R(GL) subunit from mouse. The gene covers more than 37 kb, is composed of four exons and three introns, and codes for a 1089 residue polypeptide with a calculated molecular weight of 121,000. The amino acid sequence has 60% identity with the human and rabbit R(GL). The 5' flanking region of the gene contains a TATA box, c-Myc sites, and a potential cAMP-responsive element. Muscle specific motifs, such as MyoD and MEF-2, were also found. The A-T rich 3'-UTR contained several polyadenylation signals, two associated with poly(A) down-stream consensus motifs. ARE elements, which regulate mRNA stability, were dispersed throughout the 3'-UTR. Northern analysis of poly(A) mRNA from various murine tissues indicates a major transcript of 7.5 kb in skeletal muscle and heart. Western analysis demonstrates that R(GL) protein is present in skeletal and cardiac muscle from mouse, rat, and rabbit but not in L6 myoblasts, L6 myotubes, 3T3 L1 fibroblasts, 3T3 L1 or rat primary adipocytes, confirming that expression of the gene is specific to striated muscle. Analysis of skeletal muscle from rats made diabetic by streptozotocin treatment reveals that the level of R(GL) protein is the same as in control animals, indicating that expression is not regulated by insulin.

Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL.

The regulatory-targeting subunit (RGL), also called GM) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/RGL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of RGL. To investigate the function of the phosphatase, RGL knockout mice were generated. Animals lacking RGL show no obvious defects. The RGL protein is absent from the skeletal and cardiac muscle of null mutants and present at approximately 50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by approximately 50% in the RGL-deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant RGL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by approximately 90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and RGL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that RGL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/RGL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.

Distinctive regulatory and metabolic properties of glycogen-targeting subunits of protein phosphatase-1 (PTG, GL, GM/RGl) expressed in hepatocytes.

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.

Interaction of inhibitor-2 with the catalytic subunit of type 1 protein phosphatase. Identification of a sequence analogous to the consensus type 1 protein phosphatase-binding motif.

Inhibitor-2 (I-2) is the regulatory subunit of a cytosolic type 1 Ser/Thr protein phosphatase (PP1) and potently inhibits the activity of the free catalytic subunit (CS1). Previous work from the laboratory had proposed that the interaction of I-2 with CS1 involved multiple sites (Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919-28928). The present study refines the earlier analysis and arrives at a more detailed model for the interaction between I-2 and CS1. Although the NH(2)-terminal I-2 regions containing residues 1-35 and 1-64 have no inhibitory activity on their own, they increase the IC(50) for I-2 by approximately 30-fold, indicating the presence of a CS1-interacting site. Based on several experimental approaches, we have also identified the sequence Lys(144)-Leu-His-Tyr(147) as a second site of interaction that corresponds to the RVXF motif present in many CS1-binding proteins. The peptide I-2(135-151) significantly increases the IC(50) for I-2 and attenuates CS1 inhibition. Replacement of Leu and Tyr with Ala abolishes the ability to counteract inhibition by I-2. The I-2(135-151) peptide, but not I-2(1-35), also antagonizes inhibition of CS1 by DARPP-32 in a pattern similar to that of I-2. Furthermore, a peptide derived from the glycogen-binding subunit, R(GL)/G(M)(61-80), which contains a consensus CS1-binding motif, completely counteracts CS1 inhibition by I-2 and DARPP-32. The NH(2)-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared with other interacting proteins. Other results suggest the presence of yet more sites of interaction. A model is presented in which multiple "anchoring interactions" serve to position a segment of I-2 such that it sterically occludes the catalytic pocket but need not make high affinity contacts itself.

Structure and chromosomal localization of the human glycogenin-2 gene GYG2.

Glycogenin-2 is one of two self-glucosylating proteins involved in the initiation phase of the synthesis of the storage polysaccharide glycogen. Cloning of the human glycogenin-2 gene, GYG2, has revealed the presence of 11 exons and a gene of more than 46 kb in size. The structure of the gene explains much of the observed diversity in glycogenin-2 cDNA sequences as being due to alternate exon usage. In some cases, there is variation in the splice junctions used. Over regions of protein sequence similarity, the GYG2 gene structure is similar to that of the other glycogenin gene, GYG. A genomic GYG2 clone was used to localize the gene to Xp22.3 by fluorescence in-situ hybridization. Localization close to the telomere of the short arm of the X chromosome is consistent with mapping information obtained from glycogenin-2 STS sequences. Glycogenin-2 maps between the microsatellite anchor markers AFM319te9 (DXS7100) and AFM205tf2 (DXS1060), and its 3' end is 34.5 kb from the 3' end of the arylsulphatase gene ARSD. GYG2 is outside the pseudoautosomal region PAR1 but still in a region of X-Y shared genes. As is true for several other genes in this location, an inactive remnant of GYG2, consisting of exons 1-3, may be present on the Y chromosome.

Decreased protein phosphatase 2A activity in hippocampal long-term potentiation.

Using autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) as substrate, we now find that long-term potentian (LTP) induction and maintenance are also associated with a significant decrease in calyculin A-sensitive protein phosphatase (protein phosphatase 2A) activity, without changes in Mg2+-dependent protein phosphatase (protein phosphatase 2C) activity. This decrease in protein phosphatase 2A activity was prevented when LTP induction was inhibited by treatment with calmidazolium or D-2-amino-5-phosphonopentanoic acid. In addition, the application of high-frequency stimulation to 32P-labeled hippocampal slices resulted in increases in the phosphorylation of a 55-kDa protein immunoprecipitated with anti-phosphatase 2A antibodies. Use of a specific antibody revealed that the 55-kDa protein is the B'alpha subunit of protein phosphatase 2A. Following purification of brain protein phosphatase 2A, the B'alpha subunit was phosphorylated by CaM kinase II, an event that led to the reduction of protein phosphatase 2A activity. These results suggest that the decreased activity in protein phosphatase 2A following LTP induction contributes to the maintenance of constitutively active CaM kinase II and to the long-lasting increase in phosphorylation of synaptic components implicated in LTP.

Phosphorylation-dependent inhibition of protein phosphatase-1 by G-substrate. A Purkinje cell substrate of the cyclic GMP-dependent protein kinase.

G-substrate, a specific substrate of the cGMP-dependent protein kinase, has previously been localized to the Purkinje cells of the cerebellum. We report here the isolation from mouse brain of a cDNA encoding G-substrate. This cDNA was used to localize G-substrate mRNA expression, as well as to produce recombinant protein for the characterization of G-substrate phosphatase inhibitory activity. Brain and eye were the only tissues in which a G-substrate transcript was detected. Within the brain, G-substrate transcripts were restricted almost entirely to the Purkinje cells of the cerebellum, although transcripts were also detected at low levels in the paraventricular region of the hypothalamus and the pons/medulla. Like the native protein, the recombinant protein was preferentially phosphorylated by cGMP-dependent protein kinase (Km = 0.2 microM) over cAMP-dependent protein kinase (Km = 2.0 microM). Phospho-G-substrate inhibited the catalytic subunit of native protein phosphatase-1 with an IC50 of 131 +/- 27 nM. Dephospho-G-substrate was not found to be inhibitory. Both dephospho- and phospho-G-substrate were weak inhibitors of native protein phosphatase-2A1, which dephosphorylated G-substrate 20 times faster than the catalytic subunit of protein phosphatase-1. G-substrate potentiated the action of cAMP-dependent protein kinase on a cAMP-regulated luciferase reporter construct, consistent with an inhibition of cellular phosphatases in vivo. These results provide the first demonstration that G-substrate inhibits protein phosphatase-1 and suggest a novel mechanism by which cGMP-dependent protein kinase I can regulate the activity of the type 1 protein phosphatases.

Targeted overexpression of phospholamban to mouse atrium depresses Ca2+ transport and contractility.

Phospholamban is a small phosphoprotein regulator of the Ca2+-pump of cardiac sarcoplasmic reticulum. Dephosphorylated phospholamban inhibits the Ca2+-pump and depresses contractility, whereas phosphorylation of phospholamban by cAMP-activated mechanisms relieves this inhibition and increases contractility. In order to better understand the function of phospholamban in living systems, a transgenic mouse model was established employing targeted overexpression of phospholamban to the atrium, which normally expresses low levels of the protein. Overexpression was achieved by fusing the alpha-MHC-promoter or the ANF-promoter to the phospholamban gene. Double transgenic mice were created by mating mice positive for each transgene. In single transgenic lineages, phospholamban was overexpressed four to six-fold in left atrium. In the double transgenic mice, phospholamban was overexpressed eight- to nine-fold. In the three transgenic strains. Ca2+ uptake by the sarcoplasmic reticulum was depressed to 22-30% of control values at low ionized calcium. This depression of Ca2+ uptake was largely reversed by addition of a phospholamban monoclonal antibody. In the atrial muscle strips, the time course of contraction was increased in a concentration-dependent manner by overexpression of phospholamban, whereas the basal developed tension was decreased up to 85% by phospholamban-overexpression. In all transgenic lineages, isoproterenol, a beta-adrenoceptor agonist, reversed the depression of contractility caused by overexpression of phospholamban and significantly shortened time parameters to levels approaching control values. These data demonstrate that overexpression of phospholamban in a mammalian myocardial tissue normally deficient in the protein substantially inhibits basal contractility, and furthermore suggest that in myocardial tissues containing high levels of the protein, phosphorylation of phospholamban can account for many of the positive inotropic and lusitropic effects of beta-adrenergic stimulation.

Binding of CTP:phosphocholine cytidylyltransferase to lipid vesicles: diacylglycerol and enzyme dephosphorylation increase the affinity for negatively charged membranes.

The regulation of membrane binding and activity of purified CDP:phosphocholine cytidylyl-transferase (CT) by lipid activators and enzyme dephosphorylation was examined. The binding of CT to membranes was analyzed using sucrose-loaded vesicles (SLVs). Binding to phosphatidylcholine vesicles was not detected even at a lipid:protein ratio of approximately 2000 (1 mM PC). CT bound to vesicles containing anionic lipids with apparent molar partition coefficients between 2 x 10(5) and 2 x 10(6), depending on the vesicle charge. The vesicle binding and activation of CT showed very similar sigmoidal dependencies on the lipid negative charge. In addition, diacylglycerol interacted synergistically with anionic phospholipids to stimulate both binding and activation at lower mole percent anionic lipid. These results demonstrate parallel requirements for binding and activity. Dephosphorylation of CT without destabilization was accomplished using the catalytic subunit of protein phosphatase 1. Dephosphorylated CT required a lower mole percent anionic phospholipid than phosphorylated CT for binding to and activation by SLVs. The combination of 10 mol % diacylglycerol and enzyme dephosphorylation shifted the mole percent phosphatidic acid required for half-maximal activation from 25% to 12%. These results suggest a mechanism whereby large changes in CT activity can result from changes in the phosphorylation state combined with small alterations in the membrane content of diacylglycerol. We propose a mechanism whereby dephosphorylation on the domain adjacent to the membrane binding domain increases the affinity of the latter for a negatively charged membrane surface.

Saccharomyces cerevisiae homologs of mammalian B and B' subunits of protein phosphatase 2A direct the enzyme to distinct cellular functions.

Protein phosphatase 2A (PP2A) is a major cellular serine/threonine protein phosphatase, present in the cell in a variety of heterotrimeric forms that differ in their associated regulatory B-subunit. Cloning of the mammalian B' subunit has allowed the identification of a highly homologous Saccharomyces cerevisiae gene, RTS1. Disruption of the gene results in a temperature-sensitive growth defect that can be suppressed by expression of rabbit B'alpha or B'gamma isoforms. The B'alpha subunit is much more effective in restoring normal growth at 37 degrees C than B'gamma. Immunoprecipitated Rts1p was found associated with type 2A-specific protein phosphatase activity that is sensitive to 2 nM okadaic acid, but not to 100 nM phosphatase inhibitor-2, and to be phosphorylated in vivo. However, overexpression of RTS1 was unable to suppress the cold sensitivity, defective cytokinesis, and abnormal cell morphology resulting from defects in the CDC55 gene, which encodes the yeast homolog of a different B subunit of another form of 2A phosphatase, PP2A1. These results indicate that Rts1p is a yeast homolog of the mammalian B' subunit and that the various regulatory B-subunits of PP2A are not functionally redundant but direct the enzyme to distinct cellular functions.

Dephosphorylation of Sp1 by protein phosphatase 1 is involved in the glucose-mediated activation of the acetyl-CoA carboxylase gene.

When mouse 30A5 preadipocytes are exposed to high glucose concentrations, acetyl-CoA carboxylase is induced through glucose activation of promoter II of the acetyl-CoA carboxylase gene. Glucose treatment of the cells increases Sp1 binding to two GC-rich glucose response elements in promoter II. We have investigated the mechanism by which glucose increases Sp1 binding and transactivation of promoter II in 30A5 cells. DNA mobility shift assays have shown that nuclear extracts from glucose-treated cells exhibit increased Sp1 binding activity. This increase in the binding activity is not due to glucose-mediated changes in the amount of Sp1 in the nucleus but to an increase in the activity that modifies Sp1 so that it binds more effectively to the promoter sequence. This Sp1 modifying activity is inhibited by okadaic acid and phosphatase inhibitor 2, and has a molecular mass of 38-42 kDa. The catalytic subunit of type 1 protein phosphatase, whose molecular mass is 38 kDa, also increased the ability of Sp1 to bind to promoter II. Treatment of nuclear extract with antibodies against the catalytic subunit partially suppressed the nuclear activity for Sp1 activation. From these results, we conclude that the Sp1 transcription factor exhibits enhanced binding to promoter II and transcriptional activation is the result of glucose-induced dephosphorylation by type 1 phosphatase.

Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal muscle involves mitogen-activated protein kinase-independent and rapamycin-sensitive pathways.

Incubating rat diaphragm muscles with insulin increased the glycogen synthase activity ratio (minus glucose 6-phosphate/plus glucose 6-phosphate) by approximately 2-fold. Insulin increased the activities of mitogen-activated protein (MAP) kinase and the Mr = 90,000 isoform of ribosomal protein S6 kinase (Rsk) by approximately 1.5-2.0-fold. Epidermal growth factor (EGF) was more effective than insulin in increasing MAP kinase and Rsk activity, but in contrast to insulin, EGF did not affect glycogen synthase activity. The activation of both MAP kinase and Rsk by insulin was abolished by incubating muscles with the MAP kinase kinase (MEK) inhibitor, PD 098059; however, the MEK inhibitor did not significantly reduce the effect of insulin on activating glycogen synthase. Incubating muscles with concentrations of rapamycin that inhibited activation of p70S6K abolished the activation of glycogen synthase. Insulin also increased the phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein) and promoted the dissociation of the PHAS-I*eIF-4E complex. Increasing MAP kinase activity with EGF did not mimic the effect of insulin on PHAS-I phosphorylation, and the effect of insulin on increasing MAP kinase could be abolished with the MEK inhibitor without decreasing the effect of insulin on PHAS-I. The effects of insulin on PHAS-I were attenuated by rapamycin. Thus, activation of the MAP kinase/Rsk signaling pathway appears to be neither necessary nor sufficient for insulin action on glycogen synthase and PHAS-I in rat skeletal muscle. The results indicate that the effects of insulin on increasing the synthesis of glycogen and protein in skeletal muscle, two of the most important actions of the hormone, involve a rapamycin-sensitive mechanism that may include elements of the p70S6K signaling pathway.

High complexity in the expression of the B' subunit of protein phosphatase 2A0. Evidence for the existence of at least seven novel isoforms.

Association of the catalytic subunit (C2) with a variety of regulatory subunits is believed to modulate the activity and specificity of protein phosphatase 2A (PP2A). In this study we report the cloning and expression of a new family of B-subunit, the B', associated with the PP2A0 form. Polymerase chain reactions and cDNA library screening have identified at least seven cDNA isotypes, designated alpha, beta 1, beta 2, beta 3, beta 4, gamma, and delta. The different beta subtypes appear to be generated by alternative splicing. The deduced amino acid sequences of the alpha, beta 2, beta 3, beta 4 and gamma isoforms predict molecular weights of 57,600, 56,500, 60,900, 52,500, and 68,000, respectively. The proteins are 60-80% identical and differ mostly at their termini. Two of the isoforms, B' beta 3 and B' gamma, contain a bipartite nuclear localization signal in their COOH terminus. No homology was found with other B- or B- related subunits. Northern analyses indicate a tissue-specific expression of the isoforms. Expression of B' alpha protein in Escherichia coli generated a polypeptide of approximately 53 kDa, similar to the size of the B' subunit present in the purified PP2A0. The recombinant protein was recognized by antibody raised against native B' and interacted with the dimeric PP2A (A.C2) to generate a trimeric phosphatase. The deduced amino acid sequences of the B' isoforms show significant homology to mammalian, fungal, and plant nucleotide sequences of unknown function present in the data bases. Notably, a high degree of homology (55-66%) was found with a yeast gene, RTS1, encoding a multicopy suppressor of a rox3 mutant. Our data indicate that at least seven B' subunit isoforms may participate in the generation of a large number of PP2A0 holoenzymes that may be spatially and/or functionally targeted to different cellular processes.

Phosphorylation and activation of the ATP-Mg-dependent protein phosphatase by the mitogen-activated protein kinase.

Inhibitor-2 (I-2) is the regulatory subunit of the cytosolic ATP-Mg-dependent form of type 1 serine/threonine protein phosphatase and its phosphorylation at Thr-72 by glycogen synthase kinase-3 results in phosphatase activation. Activation of cytosolic type 1 phosphatase has been observed in cells treated with growth factors. Reported here is the phosphorylation and activation of the ATP-Mg-dependent phosphatase by mitogen-activated protein kinase (MAPK). Recombinant I-2 was phosphorylated by activated MAPK to an extent (approximately 0.3 mol of phosphate/mol of polypeptide) similar to that reported for phosphorylation by the alpha isoform of glycogen synthase kinase-3. The phosphorylation of I-2 by MAPK was exclusively at Thr-72, the site involved in the activation of phosphatase. Incubation of MAPK with purified ATP-Mg-dependent phosphatase resulted in phosphorylation of the I-2 component and activation of the phosphatase. Ribosomal S6 protein kinase II (p90rsk) was also able to phosphorylate the recombinant I-2; however, this phosphorylation occurred on serines and had no effect on phosphatase activation. Our data may explain growth factor-induced activation of the ATP-Mg-dependent phosphatase and suggest that MAPK may of cytosolic type 1 phosphatase in response to insulin and/or other growth factors.

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

Casein kinase I gamma subfamily. Molecular cloning, expression, and characterization of three mammalian isoforms and complementation of defects in the Saccharomyces cerevisiae YCK genes.

Casein kinase I, one of the first protein kinases identified biochemically, is known to exist in multiple isoforms in mammals. Using a partial cDNA fragment corresponding to an isoform termed CK1 gamma, three full-length rat testis cDNAs were cloned that defined three separate members of this subfamily. The isoforms, designated CK1 gamma 1, CK1 gamma 2, and CK1 gamma 3, have predicted molecular masses of 43,000, 45,500, and 49,700. CK1 gamma 3 may also exist in an alternatively spliced form. The proteins are more than 90% identical to each other within the protein kinase domain but only 51-59% identical to other casein kinase I isoforms within this region. Messages for CK1 gamma 1 (2 kilobases (kb)), CK1 gamma 2 (1.5 and 2.4 kb), and CK1 gamma 3 (2.8 kb) were detected by Northern hybridization of testis RNA. Message for CK1 gamma 3 was also observed in brain, heart, kidney, lung, liver, and muscle whereas CK1 gamma 1 and CK1 gamma 2 messages were restricted to testis. All three CK1 gamma isoforms were expressed as active enzymes in Escherichia coli and partially purified. The enzymes phosphorylated typical in vitro casein kinase I substrates such as casein, phosvitin, and a synthetic peptide, D4. Phosphorylation of the D4 peptide was activated by heparin whereas phosphorylation of the protein substrates was inhibited. The known casein kinase I inhibitor CK1-7 also inhibited the CK1 gamma s although less effectively than the CK1 alpha or CK1 delta isoforms. All three CK1 gamma s underwent autophosphorylation when incubated with ATP and Mg2+. The YCK1 and YCK2 genes in Saccharomyces cerevisiae encode casein kinase I homologs, defects in which lead to aberrant morphology and growth arrest. Expression of mammalian CK1 gamma 1 or CK1 gamma 3 restored growth and normal morphology to a yeast mutant carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2, suggesting overlap of function between the yeast Yck proteins and these CK1 isoforms.

Domains of phosphatase inhibitor-2 involved in the control of the ATP-Mg-dependent protein phosphatase.

Inhibitor-2 (I-2) inhibits the free catalytic subunit of type 1 phosphatase (CS1) and controls the cyclic inactivation/activation of CS1 in the ATP-Mg-dependent protein phosphatase complex. We report here the effect of mutations on these two properties of I-2. Substitution of Thr-72 with Ala, Asp, or Glu generated complexes with CS1 that could not be activated. Mutation of Ser-86 did not affect activation by glycogen synthase kinase-3 (GSK-3) alone but impaired synergistic activation by casein kinase II and GSK-3. Mutations in the region between Thr-72 and Ser-86 did not alter the inhibitory potency of I-2 but prevented complete inactivation of CS1. A mutant without the 35 NH2-terminal residues exhibited an IC50 for CS1 200-fold higher than that of wild-type I-2. However, it formed an inactive phosphatase complex with CS1, which was activated by GSK-3. A mutant with the 59 COOH-terminal residues deleted retained full inhibitory activity and formed an inactive complex that could not be activated by GSK-3. We conclude that the NH2-terminal region of I-2 is involved in inhibition, that the sequence between Thr-72 and Ser-86 is necessary for the conversion of CS1 from an active to an inactive conformation, and that the COOH terminus is required for activation by GSK-3. Thus, different functional domains of I-2 may interact with distinct regions of CS1.

In vivo regulation of protein kinase C by trans-phosphorylation followed by autophosphorylation.

Dephosphorylation by the catalytic subunits of protein phosphatases 1 (CS1) and 2A (CS2) reveals that mature protein kinase C is phosphorylated at two distinct sites. Treatment of protein kinase C beta II with CS1 causes a significant increase in the protein's electrophoretic mobility (approximately 4 kDa) and a coincident loss in catalytic activity. The CS1-dephosphorylated enzyme cannot autophosphorylate or be phosphorylated by mature protein kinase C, indicating that a different kinase catalyzes the phosphorylation at this site. The loss of activity is consistent with dephosphorylation on protein kinase C's activation loop (Orr, J. W., and Newton, A. C., (1994) J. Biol. Chem. 269, 27715-27718). Treatment with CS2 results in a smaller shift in electrophoretic mobility (approximately 2 kDa) and no loss in catalytic activity. Furthermore, the CS2-dephosphorylated form can autophosphorylate and thus regain the electrophoretic mobility of mature enzyme, consistent with dephosphorylation at protein kinase C's carboxyl-terminal autophosphorylation site, which is modified in vivo (Flint, A. J., Paladini, R. D., and Koshland, D. E., Jr. (1990) Science 249, 408-411). In summary, two phosphorylations process protein kinase C to generate the mature form: a transphosphorylation that renders the kinase catalytically competent and an autophosphorylation that may be important for the subcellular localization of the enzyme.