Phosphorylase Phosphatase Activity Research Articles

A Novel Mechanism for Regulating Hepatic Glycogen Synthesis Involving Serotonin and Cyclin-Dependent Kinase-5

Hepatic autonomic nerves regulate postprandial hepatic glucose uptake, but the signaling pathways remain unknown. We tested the hypothesis that serotonin (5-hydroxytryptamine [5-HT]) exerts stimulatory and inhibitory effects on hepatic glucose disposal. Ligands of diverse 5-HT receptors were used to identify signaling pathway(s) regulating glucose metabolism in hepatocytes. 5-HT had stimulatory and inhibitory effects on glycogen synthesis in hepatocytes mediated by 5-HT1/2A and 5-HT2B receptors, respectively. Agonists of 5-HT1/2A receptors lowered blood glucose and increased hepatic glycogen after oral glucose loading and also stimulated glycogen synthesis in freshly isolated hepatocytes with greater efficacy than 5-HT. This effect was blocked by olanzapine, an antagonist of 5-HT1/2A receptors. It was mediated by activation of phosphorylase phosphatase, inactivation of glycogen phosphorylase, and activation of glycogen synthase. Unlike insulin action, it was not associated with stimulation of glycolysis and was counteracted by cyclin-dependent kinase (cdk) inhibitors. A role for cdk5 was supported by adaptive changes in the coactivator protein p35 and by elevated glycogen synthesis during overexpression of p35/cdk5. These results support a novel mechanism for serotonin stimulation of hepatic glycogenesis involving cdk5. The opposing effects of serotonin, mediated by distinct 5-HT receptors, could explain why drugs targeting serotonin function can cause either diabetes or hypoglycemia in humans.

Excess Copper‐Induced Oxidative Damages and Changes in Radish Physiology

To identify the detrimental effects of excess copper in radish (Raphanus sativus) cv. Jaunpuri, plants were grown in refined sand at control (0.001) and 0.1 and 0.2 mM (excess) copper (Cu) supplied as Cu sulphate. Previously plants were maintained for 24 days in complete nutrient solution and on the 25th day, excess Cu was superimposed. After 6 days of metal supply, the visible effects of excess Cu appeared as retardation in growth and interveinal chlorosis of young leaves; chlorosis intensified later with the development of irregular brown spots on lamina. Excess Cu not only reduced the size of leaves but also affected adversely the root development. In radish, Cu toxicity decreased the fresh weight, biomass, root weight, concentrations of total and active iron, chlorophyll, activities of antioxidative enzymes catalase (CAT), and peroxidase (POX), starch phosphorylase (ST) and acid phosphatase (AP) with concomitant increase in Cu accumulation in different parts. These effects were more pronounced at 0.2 than 0.1 mM Cu.

Biochemical Changes, Yield, and Quality of Gram Under Boron Stress

Gram (Cicer arietinum L.) cv. Avrodhi was grown in refined sand at levels of boron (B) ranging from deficiency to excess (0.0033–6.6 mg B L−1). At low (<0.33 mg B L−1) B, apart from marked depression in growth at day 28, deficiency symptoms were initiated as chlorosis of young leaflets. With increase in age, these chlorotic leaflets turned necrotic and shed premature. Biomass weight of pods and seeds was maximum at 0.33 mg B L−1. At 6.6 mg B L−1, toxicity symptoms appeared as marginal necrosis of old leaflets along with growth depression. Later, the necrotic leaflets became completely dry and shed. In low B, activity of acid phosphatase, starch phosphorylase, and ribonuclease increased and that of polyphenol oxidase decreased in leaves of gram. Excess B at 6.6 mg B L−1 increased the activity of starch phosphorylase and acid phosphatase and decreased that of ribonuclease and polyphenol oxidase. Deficiency (<0.33 mg L−1) as well as excess (>0.33 mg L−1) of B deteriorated the quality of gram seeds by lowering seed yield, starch and protein concentration, and increasing the accumulation of phenols and sugars. The concentration of B increased in leaves and seeds with an increase in B supply. The values of deficiency, threshold of deficiency, threshold of toxicity and toxicity are, respectively, 13.5, 32, 190, and 310 µg B g−1 in leaves of gram.

PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation

It has been known for over a decade that inhibition of protein phosphatase 1 (PP1) activity prevents entry into M phase, but the relevant substrate has not been identified. We report here that PP1 is required for dephosphorylation of the Cdc2-directed phosphatase Cdc25 at Ser287 (of Xenopus Cdc25; Ser216 of human Cdc25C), a site that suppresses Cdc25 during interphase. Moreover, PP1 recognizes Cdc25 directly by interacting with a PP1-binding motif in the Cdc25 N-terminus. We have also found that 14-3-3 binding to phospho-Ser287 protects Cdc25 from premature dephosphorylation. Upon entry into M phase, 14-3-3 removal from Cdc25 precedes Ser287 dephosphorylation, suggesting the existence of a phosphatase- independent pathway for 14-3-3 removal from Cdc25. We show here that this dissociation of 14-3-3 from Cdc25 requires the activity of the cyclin-dependent kinase Cdk2, providing a molecular explanation for the previously reported requirement for Cdk2 in promoting mitotic entry. Collectively, our data clarify several steps important for Cdc25 activation and provide new insight into the role of PP1 in Cdc2 activation and mitotic entry.

Validation of Interactions with Protein Phosphatase-1

This chapter presents an overview of the techniques that are available to study the interaction of protein phosphatase 1 (PP1) with other proteins. Most interactors of PP1 are inhibitors of the phosphorylase phosphatase activity of the catalytic subunit. A simple phosphatase assay can give an independent indication that the studied polypeptide is indeed an interactor of PP1. It discusses another technique known as “PP1 overlays,” based on the binding of PP1 to interactors that are associated with PVDF-membranes. PP1 overlays can be used to demonstrate a direct interaction between PP1 and a putative interactor in a complex mixture of proteins. The binding between PP1 and its interactors can also be explored by pull-down experiments when one of the two components is fused to gluthathione S-transferase (GST), a protein that binds with high affinity to glutathione-agarose. An occasional problem with GST pull-down experiments is the high background, because of the nonspecific binding of the putative PP1 interactor to GST. Another classical approach to demonstrate an interaction between two proteins is their coimmunoprecipitation from cell extracts. Some other tools are described in the chapter to study the interaction between PP1 and associated polypeptides.

Contents by Short Title

Regulation of Glc7 type 1 protein phosphatase stability and activity was studied in budding yeast. We found that the Glc7 protein has a half-life of over 180 min, which is sufficient for several generations. Glc7 protein stability was constant during the cell cycle and in batch culture growth. Furthermore, deletion of regulatory subunit Gac1, Reg1, Reg2, Sds22, or Glc8 had no influence on Glc7 protein half-life. The activity of Glc7 assayed as okadaic acid-resistant phosphorylase phosphatase activity was constant during the cell cycle. Deletion of the aforementioned regulatory subunits revealed that only Glc8 deletion had a significant effect in reducing Glc7 activity. Glc7 activity was induced during stationary phase in a Glc8-dependent manner. In addition, extracellular glucose repressed the induction of Glc7 activity. These results are consistent with glucose repression of Glc8 expression and favor the role of Glc8 as a major Glc7 activator.

Glc8 is a glucose-repressible activator of Glc7 protein phosphatase-1

Regulation of Glc7 type 1 protein phosphatase stability and activity was studied in budding yeast. We found that the Glc7 protein has a half-life of over 180 min, which is sufficient for several generations. Glc7 protein stability was constant during the cell cycle and in batch culture growth. Furthermore, deletion of regulatory subunit Gac1, Reg1, Reg2, Sds22, or Glc8 had no influence on Glc7 protein half-life. The activity of Glc7 assayed as okadaic acid-resistant phosphorylase phosphatase activity was constant during the cell cycle. Deletion of the aforementioned regulatory subunits revealed that only Glc8 deletion had a significant effect in reducing Glc7 activity. Glc7 activity was induced during stationary phase in a Glc8-dependent manner. In addition, extracellular glucose repressed the induction of Glc7 activity. These results are consistent with glucose repression of Glc8 expression and favor the role of Glc8 as a major Glc7 activator.

Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton.

The myosin phosphatase (MP) composed of the catalytic subunit of type 1 protein phosphatase and myosin phosphatase target subunit isoform 1 (MYPT1) was identified as the major serine/threonine phosphatase component in the platelet-cytoskeleton fraction. MYPT1 was phosphorylated by cytoskeletal kinase(s), but the identity of the kinase(s) and the effect of phosphorylation were not established. Incubation of platelet-cytoskeletal fraction with MgATP or MgATP[S] (magnesium adenosine 5'-[gamma-thio]triphosphate) caused a decrease in the 20 kDa light-chain of smooth-muscle myosin (MLC20) phosphatase and phosphorylase phosphatase activities. MYPT1 contains a phosphorylation site, Thr-695, involved in the inhibition of MP in a RhoA/Rho kinase-dependent manner. The cytoskeletal kinase(s) phosphorylated Thr-695 of glutathione S-transferase (GST)-MYPT1, as determined with an antibody specific for phosphorylated Thr-695. The level of Rho kinase was low in the cytoskeletal fraction and was detected primarily in the membrane and cytosolic fractions. The phosphorylation of Thr-695 by the cytoskeletal kinase(s) was not affected by Rho kinase inhibitor, Y-27632, suggesting that kinase(s) other than Rho kinase were involved. In-gel kinase assay identified a kinase at 54-59 kDa that phosphorylated the C-terminal fragment of MYPT1 (GST-MYPT1(667-1004)). Western blots detected both zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) at 54-59 kDa in the cytoskeleton and membrane fractions. Cytoskeletal ZIPK and ILK were separated and partially purified by chromatography on SP-Sepharose and on MonoQ. ZIPK preferentially phosphorylated MLC20 and had low activity on MYPT1. ILK phosphorylated both MLC20 and MYPT1 and phosphorylation of MYPT1 occured on Thr-695. The above results raise the potential for regulation of MP activity in platelet cytoskeleton by ILK and suggest an alternative to the Rho-linked pathway.

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

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.

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.

Study of the subunit interactions in myosin phosphatase by surface plasmon resonance.

The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N-terminal half (residues 1-511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11-296 > MYPT11-38 > MYPT123-38. No binding was detected with MYPT11-34, suggesting a critical role for residues 35-38, i.e. the PP1c binding motif. Binding of residues 1-22 was inferred from: a higher affinity binding to PP1c for MYPT11-38 compared to MYPT123-38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11-38, but not by MYPT123-38. Residues 40-296 (ankyrin repeats) in MYPT11-296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nM), whereas MYPT11-38, MYPT123-38 or MYPT11-34 were without effect. MYPT140-511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c-MYPT11-38 and PP1c-MYPT123-38. The inhibitory effect of MYPT140-511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11-38. The binding of MYPT1304-511 to complexes of PP1c and MYPT11-38, or MYPT11-296, was detected by SPR. These results suggest that within the N-terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c-binding motif and the other interactions are facilitated in an ordered and cooperative manner.

Neurofilament-L Is a Protein Phosphatase-1-binding Protein Associated with Neuronal Plasma Membrane and Post-synaptic Density

Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.

Phosphorylase phosphatase activity in Saccharomyces cerevisiae 257.

A phosphatase, active towards phosphorylase a and phosphorylated proteins casein and histone II-A, was isolated from Saccharomyces cerevisiae 257. The enzyme dephosphorylated glycogen phosphorylase from commercial yeast rendering it inactive. The protein phosphatase activity was not influenced by any metal ions. Phosphorylase phosphatase activity was slightly stimulated by p-nitrophenyl phosphate and inhibited by heparin.

Purification and characterisation of p99, a nuclear modulator of protein phosphatase 1 activity

We have purified a form of protein phosphatase 1 (PP1) from HeLa cell nuclei, in which the phosphatase is complexed to a regulatory subunit termed p99. We report here the cloning and characterisation of the p99 component. p99 mRNA is widely expressed in human tissues and immunofluorescence analysis with anti-p99 antibodies showed a punctate nucleoplasmic staining with additional accumulations within the nucleolus. The C-terminus of p99 contains seven RGG RNA-binding motifs, followed by eleven decapeptide repeats containing six or more of the following conserved residues (GHRPHEGPGG), and finally a putative zinc finger domain. Recombinant p99 suppresses the phosphorylase phosphatase activity of PP1 by >90% and the canonical PP1-binding motif on p99 (residues 396–401) is unusual in that the phenylalanine residue is replaced by tryptophan.

Conversion of protein phosphatase 1 catalytic subunit to a Mn(2+)-dependent enzyme impairs its regulation by inhibitor 1.

The phosphorylase phosphatase activity of protein phosphatase 1 (PP1) catalytic subunit from freshly purified rabbit skeletal muscle was inhibited by MnCl2. Prolonged storage or inhibition by nonspecific phosphatase inhibitors ATP, sodium pyrophosphate, and NaF converted the muscle PP1 to a form that required Mn2+ for enzyme activity. Recombinant PP1 catalytic subunit expressed in Escherichia coli was also a Mn2+-dependent enzyme. While native PP1 was inhibited by the phosphoprotein inhibitor I (I-1), with an IC50 of 1 nM, 40-50-fold higher concentrations of I-1 were required to inhibit the Mn2+-dependent PP1 enzymes. Conversion to the Mn2+-dependent state was accompanied by a 20-fold increase in PP1's ability to dephosphorylate and inactivate I-1. Inhibition by thiophosphorylated I-1 established that dephosphorylation does not play a significant role in I-1's reduced potency as an inhibitor of Mn2+-dependent PP1. The Mn2+-dependent PP1 enzymes were poorly inhibited by N-terminal phosphopeptides of I-1, indicating their impaired interaction with the I-1 functional domain. Mutation of a residue conserved in I-1 and DARPP-32, a structurally related PP1 inhibitor, preferentially attenuated I-1's activity as an inhibitor of Mn2+-dependent PP1. These data showed that, in addition to changes in its catalytic properties, Mn2+-dependent PP1 was modified in its interaction with I-1 at a site that was distinct from its catalytic domain. Our studies suggest that conversion to a Mn2+-dependent state alters multiple structural elements in PP1 catalytic subunit that together define its regulation by I-1.

The Interconversion of Protein Phosphatase 2A between PP2A 1and PP2A 0during Retinoic Acid-Induced Granulocytic Differentiation and a Modification on the Catalytic Subunit in S Phase of HL-60 Cells

Alterations in protein phosphatase 2A (PP2A) during retinoic acid-induced differentiation of HL-60 cells have been investigated. PP2A activity of HL-60 cells for phosphorylated myelin basic protein showed a sharp and transient increase after 18-h treatment with 1 μ mretinoic acid, which corresponded to G1/S boundary of the cell cycle. This PP2A of the 18-h treated cells was eluted from a DEAE-Sepharose column with 0.13 mNaCl, while PP2A from control cells was eluted with 0.23 mNaCl. The phosphorylase phosphatase activity of PP2A in the 0.13 meluate was greatly enhanced in the presence of protamine compared with that of the later eluting PP2A. Immunoblot analyses with antisera against B′ and Bα subunits showed that the PP2A in the 0.13 mNaCl eluate from 18-h retinoic acid-treated cells was PP2A 0(AC-B′), whereas the PP2A eluted with 0.23 mNaCl from 24-h retinoic acid-treated cells and 0-, 18-, and 24-h control cells was PP2A 1(AC-Bα). These results strongly suggest that PP2A undergoes a transient and reversible interconversion of holoenzyme forms during the initial stage of retinoic acid-induced granulocytic differentiation. PP2A activity assayed after dissociation of the catalytic subunit, for phosphorylase as substrate, showed a sharp and transient decrease in S phase of HL-60 cells irrespective of the presence or absence of retinoic acid. Immunoblot analyses with antisera against C-terminus and N-terminus of the catalytic subunit of PP2A suggested that a modification at the C-terminus is responsible for the decrease in PP2A activity. Immunoreactivity to the C-terminal antibody was restored after treatments of the S-phase extract with alkali or ethanol, the conditions which remove the methyl group from the C-terminus. These results suggest that the C-terminus of PP2A catalytic subunit is transiently methylated in S phase of HL-60 cells.

High Molecular Weight Protein Phosphatase Type 1 Dephosphorylates the Retinoblastoma Protein

pRb controls cell proliferation by restricting inappropriate entry of cells into the cell division cycle. As dephosphorylation of pRb during mitotic exit activates its growth suppressive function, identification of the protein phosphatase that dephosphorylates pRb, and characterization of the mechanism of its regulation, are essential to elucidating the mechanisms of cell growth control. By fractionating mitotic CV-1P cell extracts, we identify the protein phosphatase which dephosphorylates pRb as a type 1 serine/threonine phosphoprotein phosphatase (PP1). Molecular sizing analyses indicate that the catalytic enzyme (PP1c) is present in a high molecular weight complex, with a predicted molecular mass of 166 kDa. PP1-interacting proteins in the mitotic cell extracts are identified. Two PP1-interacting proteins (41 and 110 kDa) are shown to form distinct complexes with PP1c from fractions of separated mitotic cell extracts containing phosphorylase phosphatase activity. However, only the 110-kDa PP1-interacting protein is present in fractions containing pRb-directed phosphatase activity, identifying this protein as a putative activator of PP1 function toward pRb during mitosis.

Further evidence that inhibitor-2 acts like a chaperone to fold PP1 into its native conformation

The γ 1-isoform of protein phosphatase-1 expressed in Escherichia coli (PP1γ) and the native PP1 catalytic subunit (PP1C) isolated from skeletal muscle dephosphorylated Ser-14 of glycogen phosphorylase at comparable rates. In contrast, PP1γ dephosphorylated several tyrosine-phosphorylated proteins at similar rates to authentic protein tyrosine phosphatases (PTPases), but native PP1C was almost inactive towards these substrates. The phosphorylase phosphatase (PhP) and PTPase activities of PP1γ were inhibited by vanadate with IC 50 values (30–100 μM) comparable to authentic PTPases, whereas the PhP activity of native PP1C was insensitive to vanadate. PP1γ lost its PTPase activity, and its PhP activity became insensitive to vanadate, after interaction with inhibitor-2, followed by the reversible phosphorylation of inhibitor-2 at Thr-72. These findings support and extend the hypothesis that inhibitor-2 functions like a chaperone to fold PP1 into its native conformation, and suggest that the correct folding of PP1 may be critical to prevent the uncontrolled dephosphorylation of cellular phosphotyrosine residues.