Catalytic Subunit Of Protein Phosphatase Research Articles

Crystal Structure of the Complex between Calyculin A and the Catalytic Subunit of Protein Phosphatase 1

The crystal structure of the catalytic subunit of the protein phosphatase 1 (PP1), PP1γ, in complex with a marine toxin, calyculin A, was determined at 2.0 Å resolution. The metal binding site contains the phosphate group of calyculin A and forms a tight network via the hydrophilic interactions between PP1 and calyculin A. Calyculin A is located in two of the three grooves, namely, in the hydrophobic groove and the acidic groove on the molecular surface. This is the first observation to note that the inhibitor adopts not a pseudocyclic conformation but an extended conformation in order to form a complex with the protein. The amino acid terminus of calyculin A contributes, in a limited manner, to the binding to PP1γ, which is consistent with findings from the studies of dose-inhibition analysis.

Tight Binding Inhibition of Protein Phosphatase-1 by Phosphatidic Acid: SPECIFICITY OF INHIBITION BY THE PHOSPHOLIPID

Phosphatidic acid (PA) has been identified as a bioactive lipid second messenger, yet despite extensive investigation, no cellular target has emerged as a mediator of its described biological effects. In this study, we identify the gamma isoform of the human protein phosphatase-1 catalytic subunit (PP1c gamma) as a high affinity in vitro target of PA. PA inhibited the enzyme dose-dependently with an IC(50) of 15 nm. Mechanistically, PA inhibited the enzyme noncompetitively with the kinetics of a tight binding inhibitor and a K(i) value of 0.97 +/- 0.24 nm. Together, these data describe one of the most potent in vitro effects of PA. To further elucidate the interaction between PA and PP1c gamma, structure/function analysis of the lipid was carried out using commercially available and synthetically generated analogs of PA. These studies disclosed that the lipid-protein interaction is dependent on the presence of the lipid phosphate as well as the presence of the fatty acid side chains, because lipids lacking either of these substituents resulted in complete loss of inhibition. However, the specific composition of the fatty acid side chains was not important for inhibition. Using 1-O-hexadecyl,2-oleoyl-PA, it was also shown that the carbonyl group of the sn-1 acyl linkage is not required for the lipid-protein interaction. Finally, using a lipid-protein overlay assay, it was demonstrated that PP1c gamma specifically and directly interacts with phosphatidic acid while not significantly binding other phospholipids. These results identify PA as a tight binding and specific inhibitor of PP1, and they raise the hypothesis that PP1c gamma may function as a mediator of PA action in cells. They also argue for the existence of a specific high affinity PA-binding domain on the enzyme.

Essential functions of Sds22p in chromosome stability and nuclear localization of PP1.

Sds22p is a conserved, leucine-rich repeat protein that interacts with the catalytic subunit of protein phosphatase 1 (PP1(C)) and which has been proposed to regulate one or more functions of PP1(C) during mitosis. Here we show that Saccharomyces cerevisiae Sds22p is a largely nuclear protein, most of which is present as a sTable 1:1 complex with yeast PP1(C) (Glc7p). Temperature-sensitive (Ts(-)) S. cerevisiae sds22 mutants show profound chromosome instability at elevated growth temperatures but do not confer a cell cycle stage-specific arrest. In the sds22-6 Ts(-) mutant, nuclear Glc7p is both reduced in level and aberrantly localized at 37 degrees C and the interaction between Glc7p and Sds22p in vitro is reduced at higher temperatures, consistent with the in vivo Ts(-) growth defect. Like some glc7 mutations, sds22-6 can suppress the Ts(-) growth defect associated with ipl1-2, a loss of function mutation in a protein kinase that is known to work in opposition to PP1 on at least two nuclear substrates. This, together with reciprocal genetic interactions between GLC7 and SDS22, suggests that Sds22p functions positively with Glc7p to promote dephosphorylation of nuclear substrates required for faithful transmission of chromosomes during mitosis, and this role is at least partly mediated by effects of Sds22p on the nuclear distribution of Glc7p

Purification and characterization of protein phosphatase-1 from two cold-hardy goldenrod gall insects.

The catalytic subunit of protein phosphatase-1 (PP-1) was purified to homogeneity from final instar larvae (the overwintering stage) of freeze avoiding (Epiblema scudderiana) and freeze tolerant (Eurosta solidaginis) cold-hardy insects. Arrhenius plots showed that activity of PP-1 from both species was strongly suppressed at low temperature. Acidic shifts in pH optima and increased inhibition by okadaic acid were also observed when the enzymes were assayed at 4 degrees C compared with 24 degrees C. The data identify multiple ways by which PP-1 can be inhibited at low temperature and this inhibition appears to be key to sustaining high glycogen phosphorylase activity in support of polyol synthesis at low temperatures.

The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats.

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, G(L), R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, G(L)-PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and G(L) complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and G(L) were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6-PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and G(L) and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for G(L). The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of G(L)-PP1c, suggesting that R5-PP1c may function as a hepatic phosphorylase phosphatase, whereas G(L)-PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and G(L)

The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, GL, R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, GL–PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and GL complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and GL were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6–PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and GL and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for GL. The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of GL–PP1c, suggesting that R5–PP1c may function as a hepatic phosphorylase phosphatase, whereas GL–PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and GL

Purification and properties of Arabidopsis thaliana type 1 protein phosphatase (PP1)

The Arabidopsis thaliana type 1 protein phosphatase (PP1) catalytic subunit was released from its endogenous regulatory subunits by ethanol precipitation and purified by anion exchange and microcystin affinity chromatography. The enzyme was identified by MALDI-TOF mass spectrometry from a tryptic digest of the purified protein as a mixture of PP1 isoforms (TOPP 1–6) indicating that at least 4–6 of the eight known PP1 proteins are expressed in sufficient quantities for purification from A. thaliana suspension cells. The enzyme had a final specific activity of 8950 mU/mg using glycogen phosphorylase a as substrate, had a subunit molecular mass of 35 kDa as determined by SDS–PAGE and behaved as a monomeric protein of approx. 39 kDa on Superose 12 gel filtration chromatography. Similar to the mammalian type 1 protein phosphatases, the A. thaliana enzyme was potently inhibited by Inhibitor-2 (IC 50=0.65 nM), tautomycin (IC 50=0.06 nM), microcystin-LR (IC 50=0.01 nM), nodularin (IC 50=0.035 nM), calyculin A (IC 50=0.09 nM), okadaic acid (IC 50=20 nM) and cantharidin (IC 50=60 nM). The enzyme was also inhibited by fostriecin (IC 50=22 μM), NaF (IC 50=2.1 mM), Pi (IC 50=9.5 mM), and PPi (IC 50=0.07 mM). Purification of the free catalytic subunit allowed it to be used to probe protein phosphatase holoenzyme complexes that were enriched on Q-Sepharose and a microcystin-Sepharose affinity matrix and confirmed several proteins to be PP1 targeting subunits.

Myr 8, A Novel Unconventional Myosin Expressed during Brain Development Associates with the Protein Phosphatase Catalytic Subunits 1α and 1γ1

Directed neuronal, astroglial, and oligodendroglial cell migrations comprise a prominent feature of mammalian brain development. Because molecular motor proteins have been implicated in a wide spectrum of processes associated with cell motility, we initiated studies to define the pool of myosins in migrating cerebellar granule neurons and type-1 neocortical astrocytes. Our analyses identified two isoforms of a novel unconventional myosin, which we have cloned, sequenced, and designated myr 8a and 8b (eighth unconventional myosin from rat). Phylogenetic analysis indicates that myr 8 myosins comprise a new class of myosins, which we have designated class XVI. The head domain contains a large N-terminal extension composed of multiple ankyrin repeats, which are implicated in mediating an association with the protein phosphatase 1 (PP1) catalytic subunits 1alpha and 1gamma. The motor domain is followed by a single putative light-chain binding domain. The tail domain of myr 8a is comparatively short with a net positive charge, whereas the tail domain of myr 8b is extended, bears an overall neutral charge, and reveals several stretches of poly-proline residues. Neither the myr 8a nor the myr 8b sequence reveals alpha-helical coiled-coil motifs, suggesting that these myosins exist as monomers. Both immunoblot and Northern blot analyses indicate that myr 8b is the predominant isoform expressed in brain, principally at developmental time periods. The structural features and restricted expression patterns suggest that members of this novel class of unconventional myosins comprise a mechanism to target selectively the protein phosphatase 1 catalytic subunits 1alpha and/or 1gamma in developing brain.

Cell-cycle-dependent association of protein phosphatase 1 and focal adhesion kinase.

Immunofluorescence studies with protein phosphatase-1 (PP1) isoforms-specific antibodies detected PP1delta, but not alpha or gamma1, at focal adhesions. PP1delta also co-immunoprecipitated with the focal adhesion kinase (FAK) and the alphav-integrin. In the present study glutathione S-transferase (GST)-PP1delta pulled-down FAK from fibroblasts extract and the interaction domain localized between residues 159 and 295 of delta. The association was confirmed by the ability to GST-FAK-related non-kinase (FRNK) to pull-down PP1delta from fibroblasts extract. GST-FRNK also pulled-down purified muscle PP1 catalytic subunit, thus indicating direct interaction between FAK and PP1. FAK displays consensus sequences for phosphorylation by cell division cycle kinase-2-cyclin B, and might be a PP1 substrate. In fact, FAK immunoprecipitated from metabolically-labelled mitotic HeLa cells without tyrosine phosphatase inhibitors was phosphorylated on Ser only and was dephosphorylated in vitro by purified muscle PP1, with loss of phospho-Ser. No PP1 was associated with FAK immunoprecipitated from mitotic HeLa cells. However, progressively more PP1 activity was assayed in FAK-immunoprecipitates obtained from cells released from mitosis. The associated activity was maximal at 2 h from the mitotic release (when 85-90% of the cells remained round) and decreased to basal level by 8 h (when cells were all polygonal). At the same time FAK underwent dephosphorylation, which was completed by 4 h. FAK obtained from cells at 1.5 h was Ser-phosphorylated, and underwent dephosphorylation during in vitro incubation, with loss of phospho-Ser, indicating the presence of active FAK-bound phosphatase. The only FAK-associated PP1 isoform between 1 and 8 h was PP1delta. The results suggest that FAK dephosphorylation by PP1delta occurs in cells released from mitosis, and confirmed the specific association of PP1delta, as detected previously in adherent cells.

Cell-cycle-dependent association of protein phosphatase 1 and focal adhesion kinase

Immunofluorescence studies with protein phosphatase-1 (PP1) isoforms-specific antibodies detected PP1δ, but not α or γ1, at focal adhesions. PP1δ also co-immunoprecipitated with the focal adhesion kinase (FAK) and the αv-integrin. In the present study glutathione S-transferase (GST)–PP1δ pulled-down FAK from fibroblasts extract and the interaction domain localized between residues 159 and 295 of δ. The association was confirmed by the ability to GST–FAK-related non-kinase (FRNK) to pull-down PP1δ from fibroblasts extract. GST–FRNK also pulled-down purified muscle PP1 catalytic subunit, thus indicating direct interaction between FAK and PP1. FAK displays consensus sequences for phosphorylation by cell division cycle kinase-2–cyclin B, and might be a PP1 substrate. In fact, FAK immunoprecipitated from metabolically-labelled mitotic HeLa cells without tyrosine phosphatase inhibitors was phosphorylated on Ser only and was dephosphorylated in vitro by purified muscle PP1, with loss of phospho-Ser. No PP1 was associated with FAK immunoprecipitated from mitotic HeLa cells. However, progressively more PP1 activity was assayed in FAK-immunoprecipitates obtained from cells released from mitosis. The associated activity was maximal at 2h from the mitotic release (when 85–90% of the cells remained round) and decreased to basal level by 8h (when cells were all polygonal). At the same time FAK underwent dephosphorylation, which was completed by 4h. FAK obtained from cells at 1.5h was Ser-phosphorylated, and underwent dephosphorylation during in vitro incubation, with loss of phospho-Ser, indicating the presence of active FAK-bound phosphatase. The only FAK-associated PP1 isoform between 1 and 8h was PP1δ. The results suggest that FAK dephosphorylation by PP1δ occurs in cells released from mitosis, and confirmed the specific association of PP1δ, as detected previously in adherent cells.

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity.

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2alpha kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2alpha kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA.Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho- and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2alpha.

Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2alpha.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) on serine 51 integrates general translation repression with activation of stress-inducible genes such as ATF4, CHOP, and BiP in the unfolded protein response. We sought to identify new genes active in this phospho-eIF2alpha-dependent signaling pathway by screening a library of recombinant retroviruses for clones that inhibit the expression of a CHOP::GFP reporter. A retrovirus encoding the COOH terminus of growth arrest and DNA damage gene (GADD)34, also known as MYD116 (Fornace, A.J., D.W. Neibert, M.C. Hollander, J.D. Luethy, M. Papathanasiou, J. Fragoli, and N.J. Holbrook. 1989. Mol. Cell. Biol. 9:4196-4203; Lord K.A., B. Hoffman-Lieberman, and D.A. Lieberman. 1990. Nucleic Acid Res. 18:2823), was isolated and found to attenuate CHOP (also known as GADD153) activation by both protein malfolding in the endoplasmic reticulum, and amino acid deprivation. Despite normal activity of the cognate stress-inducible eIF2alpha kinases PERK (also known as PEK) and GCN2, phospho-eIF2alpha levels were markedly diminished in GADD34-overexpressing cells. GADD34 formed a complex with the catalytic subunit of protein phosphatase 1 (PP1c) that specifically promoted the dephosphorylation of eIF2alpha in vitro. Mutations that interfered with the interaction with PP1c prevented the dephosphorylation of eIF2alpha and blocked attenuation of CHOP by GADD34. Expression of GADD34 is stress dependent, and was absent in PERK(-)/- and GCN2(-)/- cells. These findings implicate GADD34-mediated dephosphorylation of eIF2alpha in a negative feedback loop that inhibits stress-induced gene expression, and that might promote recovery from translational inhibition in the unfolded protein response.

Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1

To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns > 99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1–46.5cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related ∼ 110–130kDa MYPT subunits by its molecular mass of 58kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)–MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST–MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.

Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1.

To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns >99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1-46.5 cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529 Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related approximately 110-130 kDa MYPT subunits by its molecular mass of 58 kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)-MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST-MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.

Multiple Interactions within the AKAP220 Signaling Complex Contribute to Protein Phosphatase 1 Regulation

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (K(i) = 2.9 +/- 0.7 micrometer). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.

The C-terminus of NIPP1 (nuclear inhibitor of protein phosphatase-1) contains a novel binding site for protein phosphatase-1 that is controlled by tyrosine phosphorylation and RNA binding

Nuclear inhibitor of protein phosphatase-1 (NIPP1; 351 residues) is a nuclear RNA-binding protein that also contains in its central domain two contiguous sites of interaction with the catalytic subunit of protein phosphatase-1 (PP1(C)). We show here that mutation of these phosphatase-interaction sites did not completely abolish the ability of NIPP1 to bind and inhibit PP1(C). This could be accounted for by an additional inhibitory phosphatase-binding site in the C-terminal region (residues 311-351), with an inhibitory core corresponding to residues 331-337. Following mutation of all three PP1(C)-binding sites in the central and C-terminal domains, NIPP1 no longer interacted with PP1(C). Remarkably, while both NIPP1 domains inhibited the phosphorylase phosphatase activity of PP1(C) independently, mutation of either domain completely abolished the ability of NIPP1 to inhibit the dephosphorylation of myelin basic protein. The inhibitory potency of the C-terminal site of NIPP1 was decreased by phosphorylation of Tyr-335 and by the addition of RNA. Tyr-335 could be phosphorylated by tyrosine kinase Lyn, but only in the presence of RNA. In conclusion, NIPP1 contains two phosphatase-binding domains that function co-operatively but which are controlled independently. Our data are in agreement with a shared-site model for the interaction of PP1(C) with its regulatory subunits.

Glycogen Synthase Association with the Striated Muscle Glycogen-targeting Subunit of Protein Phosphatase-1: SYNTHASE ACTIVATION INVOLVES SCAFFOLDING REGULATED BY β-ADRENERGIC SIGNALING

Glycogen-binding subunits for protein phosphatase-1 (PP1) target the PP1 catalytic subunit (PP1C) to glycogen particles, where the enzymes glycogen synthase and glycogen phosphorylase are concentrated. Here we identify sites within the striated muscle glycogen-binding subunit (G(M)) that mediate direct binding to glycogen synthase. Both PP1C and glycogen synthase were coimmunoprecipitated with a full-length FLAG-tagged G(M) transiently expressed in COS7 cells or C2C12 myotubes. Deletion and mutational analysis of a glutathione S-transferase (GST) fusion of the N-terminal domain of G(M) (residues 1-240) identified two putative sites for binding to glycogen synthase, one of which is the WXNXGXNYX(I/L) motif that is conserved among the family of PP1 glycogen-binding subunits. Either deletion of this motif or Ala substitution of Asn-228 in this motif disrupted the binding of glycogen synthase. Expression of full-length FLAG-G(M) in cells increased the activity of endogenous glycogen synthase, but protein disabled in either PP1 binding or glycogen synthase binding did not produce synthase activation. The results show that efficient activation of glycogen synthase requires a scaffold function of G(M) that involves simultaneous binding of both PP1C and glycogen synthase. Isoproterenol and forskolin treatment of cells decreased glycogen synthase binding to FLAG-G(M), thereby limiting synthase activation by PP1. This response was insensitive to inhibition by H-89, therefore probably not involving cAMP-dependent protein kinase, but did require inclusion of microcystin-LR during cell lysis, implying that phosphorylation was modulating binding of glycogen synthase. Phosphorylation control of binding to a scaffold site on the G(M) subunit of PP1 offers a new mechanism for regulation of muscle glycogen synthase in response to beta-adrenergic signals.

Glc7p protein phosphatase inhibits expression of glutamine-fructose-6-phosphate transaminase from GFA1.

Inhibitor-1 (I-1) is a specific inhibitor of protein phosphatase-1 (PP1). We assayed the ability of I-1 to inhibit Saccharomyces cerevisiae PP1, Glc7p, in vivo. Glc7p like other PP1 catalytic subunits associates with a variety of noncatalytic subunits, and Glc7p holoenzymes perform distinct physiological roles. Our results show that I-1 inhibits Glc7p holoenzymes that regulate transcription and mitosis, but holoenzymes responsible for meiosis and glycogen metabolism were unaffected. Additionally, we exploited a genetic screen for mutants that were dependent on I-1 to grow. This scheme can identify processes that are negatively regulated by Glc7p-catalyzed dephosphorylation. In this paper I-1-dependent gfa1 mutations were analyzed in detail. GFA1 encodes glutamine-fructose-6-phosphate transaminase. One or more phosphorylated proteins activate GFA1 transcription because the pheromone response and Pkc1p/mitogen-activated protein kinase pathways positively regulate GFA1 transcription. Our findings show that an I-1-sensitive Glc7p holoenzyme reduces GFA1 transcription. Therefore, GFA1 is a member of a growing list of genes that are negatively regulated by Glc7p dephosphorylation.

Sds22p Is a Subunit of a Stable Isolatable Form of Protein Phosphatase 1 (Glc7p) from Saccharomyces cerevisiae

Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. PP1 activity is believed to be controlled by the interaction of PP1 catalytic subunit with various regulatory subunits. The essential gene GLC7 encodes the PP1 catalytic subunit in Saccharomyces cerevisiae. In this study, full-length GLC7(1–312), C-terminal deletion mutants, and C-terminally poly-his tagged mutants were constructed and expressed in a GLC7 knockout strain of S. cerevisiae. Viability studies of the GLC7 knockout strains carrying the plasmids expressing GLC7 C-terminal deletion mutants and their tagged forms showed that the mutants 1–295 and 1–304 were functional, whereas the mutant 1–245 was not. The C-terminally poly-his tagged Glc7p with and without an N-terminal hemagglutinin (HA) tag was partially purified by immobilized Ni2+ affinity chromatography and further analyzed by gel filtration and ion exchange chromatography. Phosphatase activity assays, SDS–PAGE, and Western blot analyses of the chromatographic fractions suggested that the Glc7p associated with regulatory subunits in vivo. A 40-kDa protein was copurified with tagged Glc7p through several chromatographic procedures. Monoclonal antibody against the HA tag coimmunoprecipitated the tagged Glc7p and the 40-kDa protein. This protein was further purified by a reverse phase HPLC column. Analysis by CNBr digestion, peptide sequencing, and electrospray mass spectrometry showed that this 40-kDa protein is Sds22p, one of the proteins proposed to be a regulatory subunit of Glc7. These results demonstrate that Sds22p forms a complex with Glc7p and that Sds22p:Glc7p is a stable isolatable form of yeast PP1.

Protein Phosphatase 2A Inhibitors, I1PP2A and I2PP2A, Associate with and Modify the Substrate Specificity of Protein Phosphatase 1

Recombinant I(1)(PP2A) and I(2)(PP2A) did not affect the activity of the catalytic subunit of protein phosphatase 1 (PP1(C)) with (32)P-labeled myelin basic protein, histone H1, and phosphorylase when assayed in the absence of divalent cations. However, in the presence of Mn(2+), I(1)(PP2A) and I(2)(PP2A) stimulated PP1(C) activity by 15-20-fold with myelin basic protein and histone H1 but not phosphorylase. Half-maximal stimulation occurred at 2 and 4 nM I(1)(PP2A) and I(2)(PP2A), respectively. Moreover, I(1)(PP2A) and I(2)(PP2A) reduced the Mn(2+) requirement by about 30-fold to 10 microM. In contrast, PP1(C) activity was unaffected by I(1)(PP2A) and I(2)(PP2A) in the presence of Co(3+) (0.1 mM), Mg(2+) (2 mM), Ca(2+) (0.5 mM), and Zn(2+) (0.1 mM). Following gel filtration chromatography on Sephacryl S-200 in the presence of Mn(2+), PP1(C) coeluted with I(1)(PP2A) and I(2)(PP2A) in the void volume. However, when I(1)(PP2A) and I(2)(PP2A) or Mn(2+) were omitted, PP1(C) emerged with a V(e)/V(0) of approximately 1.6. The results demonstrate that I(1)(PP2A) and I(2)(PP2A) associate with and modify the substrate specificity of PP1(C) in the presence of physiological concentrations of Mn(2+). A novel role is suggested for I(1)(PP2A) and I(2)(PP2A) in the reciprocal regulation of two major mammalian serine/threonine phosphatases, PP1 and PP2A.