The structure, role, and regulation of type 1 protein phosphatases.

Type 1 protein phosphatases (PP-1) comprise a group of widely distributed enzymes that specifically dephosphorylate serine and threonine residues of certain phosphoproteins. They all contain an isoform of the same catalytic subunit, which has an extremely conserved primary structure. One of the properties of PP-1 that allows one to distinguish them from other serine/threonine protein phosphatases is their sensitivity to inhibition by two proteins, termed inhibitor 1 and inhibitor 2, or modulator. The latter protein can also form a 1:1 complex with the catalytic subunit that slowly inactivates upon incubation. This complex is reactivated in vitro by incubation with MgATP and protein kinase FA/GSK-3. In the cell the type 1 catalytic subunit is associated with noncatalytic subunits that determine the activity, the substrate specificity, and the subcellular location of the phosphatase. PP-1 plays an essential role in glycogen metabolism, calcium transport, muscle contraction, intracellular transport, protein synthesis, and cell division. The activity of PP-1 is regulated by hormones like insulin, glucagon, alpha- and beta-adrenergic agonists, glucocorticoids, and thyroid hormones.     

Involvement of protein phsophatase-1 in cytoskeletal organization of cultured endothelial cells

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Crystal structure and mutagenesis of a protein phosphatase-1:calcineurin hybrid elucidate the role of the beta12-beta13 loop in inhibitor binding

Protein phosphatase-1 and protein phosphatase-2B (calcineurin) are eukaryotic serine/threonine phosphatases that share 40% sequence identity in their catalytic subunits. Despite the similarities in sequence, these phosphatases are widely divergent when it comes to inhibition by natural product toxins, such as microcystin-LR and okadaic acid. The most prominent region of non-conserved sequence between these phosphatases corresponds to the beta12-beta13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin. In the present study, mutagenesis of residues 273-277 of the beta12-beta13 loop of the protein phosphatase-1 catalytic subunit (PP-1c) to the corresponding residues in calcineurin (312-316), resulted in a chimeric mutant that showed a decrease in sensitivity to microcystin-LR, okadaic acid, and the endogenous PP-1c inhibitor protein inhibitor-2. A crystal structure of the chimeric mutant in complex with okadaic acid was determined to 2.0-A resolution. The beta12-beta13 loop region of the mutant superimposes closely with that of wild-type PP-1c bound to okadaic acid. Systematic mutation of each residue in the beta12-beta13 loop of PP-1c showed that a single amino acid change (C273L) was the most influential in mediating sensitivity of PP-1c to toxins. Taken together, these data indicate that it is an individual amino acid residue substitution and not a change in the overall beta12-beta13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid.     

The isolation of novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus nuclei.

Nuclei from bovine thymus contain a high level of partially latent protein phosphatase 1 (PP-1). More than 90% of this PP-1 is associated with the insoluble chromatin/matrix fraction and can be extracted with 0.3 M NaCl. The salt extract also contains three heat- and acid-stable inhibitory proteins of PP-1 that can be resolved on Mono Q. We have purified two of these nuclear inhibitors of PP-1 (NIPP-1a and NIPP-1b) until homogeneity. They are acidic proteins (pI = 4.4) with a molecular mass of 18 kDa (NIPP-1a) and 16 kDa (NIPP-1b) on SDS-PAGE. Judged from the larger molecular mass that was deduced from gel filtration (35 kDa), NIPP-1a and NIPP-1b appear to be asymmetric or dimeric proteins. The nuclear inhibitors totally inhibited the phosphorylase phosphatase activity of PP-1, but even at a 250-fold higher concentration they did not affect the activities of the other major serine/threonine protein phosphatases (PP-2A, PP-2B, and PP-2C). NIPP-1a and NIPP-1b inhibited the catalytic subunit of PP-1 with an extrapolated Ki of about 1 pM, which is some three orders of magnitude better than the cytoplasmic proteins inhibitor 1/DARPP-32 and modulator. The nuclear inhibitors were not inactivated by incubation with protein phosphatases that inactivate inhibitor 1 and DARPP-32. Unlike modulator, they were not able to convert the catalytic subunit of PP-1 into a MgATP-dependent form. Remarkably, the extent of inhibition of PP-1 by NIPP-1b depended on the nature of the substrate. The phosphorylase phosphatase and casein phosphatase activities of PP-1 were completely blocked by NIPP-1b, whereas the dephosphorylation of basic proteins was either not at all inhibited (histone IIA) or only partially (myelin basic protein). These data may indicate that the acidic NIPP-1b is inactivated through complexation by basic proteins. Indeed, nonphosphorylated histone IIA antagonized the inhibitory effect of NIPP-1b on the casein phosphatase activity of PP-1. Our data show that the nucleus contains specific and potent inhibitory proteins of PP-1 that differ from earlier described cytoplasmic inhibitors. We suggest that these novel proteins may control the activity of nuclear PP-1 on its natural substrate(s).     

Protein phosphatases possibly involved in rat spermatogenesis

The expression of mRNAs for catalytic subunits of serine/threonine protein phosphatases 1 (PP-1) and 2A (PP-2A) in various rat tissues were examined. Four kinds of probes were used to detect mRNAs for two isotypes of PP-1 (dis2m1 and dis2m2), and two isotypes of PP-2A (PP-2A alpha and PP-2A beta). mRNAs for all of these four catalytic subunits were expressed in almost all tissues at substantial levels. They were expressed in two different sizes in most tissues. Remarkable evidence is that the smaller sized mRNAs of dis2m1 and PP-2A beta, 1.8 kb and 1.4 kb in length, respectively, were specifically highly expressed in testis. Both these smaller sized mRNAs began to be expressed at the age when meiosis started and were detected in testicular germ cells at the pachytene stage of meiotic prophase. Protein phosphatases which have peptides encoded by dis2m1 and PP-2A beta as catalytic subunits may play important roles in spermatogenesis.     

Alpha4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases

The catalytic activity of the C subunit of serine/threonine phosphatase 2A is regulated by the association with A (PR65) and B subunits. It has been reported that the alpha4 protein, a yeast homolog of the Tap42 protein, binds the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases such as protein phosphatase 4 and protein phosphatase 6. In the present study, we showed that alpha4 binds these three phosphatases and the association of alpha4 reduces the activities of these phosphatases in vitro. In contrast, PR65 binds to the C subunit of serine/threonine phosphatase 2A but not to protein phosphatase 4 and protein phosphatase 6. These results suggest that the alpha4 protein is a common regulator of the C subunit of serine/threonine phosphatase 2A and protein phosphatase 2A-related protein phosphatases.     

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

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

Identification of sds21 in fission yeast in an inhibitor-resistant high molecular mass protein phosphatase-1 complex.

While characterizing the type-1 protein phosphatases sds21 and dis2 in fission yeast (Schizosaccharomyces pombe) a novel high molecular mass protein was identified with serine/threonine phosphatase activity (referred to as PP-R) that was resistant to a panel of characteristic inhibitors of protein phosphatases. Purification of the native sds21 catalytic isoform of protein phosphatase-1 (PP-1) from an S. pombe knockout strain lacking dis2 (deltadis2) resulted predominantly in identification of PP-R. To test the hypothesis that the catalytic activity of PP-R comprised sds21, a parallel purification was performed of PP-1 activity from an S. pombe knockout strain lacking sds21 (deltasds21). Both deltasds21 and deltadis2 strains exhibited similar protein phosphatase activity profiles as determined by DEAE-sepharose, Mono-Q and Superdex gel filtration chromatography. However, the peak of protein phosphatase activity from deltasds21 S. pombe that co-migrated with PP-R from deltadis2 S. pombe exhibited the sensitivity to a panel of inhibitors that was characteristic of a type-1 protein phosphatase. These data suggest that the catalytic subunit of PP-R comprises sds21 and that the resistance to inhibitors may originate from structural differences between dis2 and sds21 isoforms. A key structural feature present in sds21, but lacking in dis2, is a classical phosphorylation consensus sequence surrounding serine-145 of sds21. The previous hypothesis was that PP-1 activity among several lower eukaryotes may be regulated directly by cAMP-dependent protein kinase (PKA) phosphorylation. However, this study demonstrated that recombinant sds21 is not a target for PKA in vitro. The constrained configuration of the putative PKA site on the PP-1 holoenzyme may restrict its ability to be targeted by PKA.     

Design and synthesis of AX7574: a microcystin-derived, fluorescent probe for serine/threonine phosphatases

The design and synthesis of AX7574, a microcystin-derived probe for serine/threonine phosphatases, is described. A key step in the synthesis was the conjugation under basic conditions of a tetramethylrhodamine 1,3-diketone derivative to the arginine side chain present in microcystin-LR. The resulting conjugate specifically labeled the active site of protein phosphatases 1 (PP-1) with a 1:1 stoichiometry and IC50 of 4.0 nM. AX7574 was used to isolate and identify PP-1, PP-2A, PP-4, and PP-6 in Jurkat cells. Finally, AX7574 was able to record changes in the phosphatase activity levels of calyculin A treated Jurkat cells versus untreated control cells.     

Regulation of protein phosphatase-1G from rabbit skeletal muscle. 1. Phosphorylation by cAMP-dependent protein kinase at site 2 releases catalytic subunit from the glycogen-bound holoenzyme

The glycogen-associated form of protein phosphatase-1 (PP-1G) is a heterodimer comprising a 37-kDa catalytic (C) subunit and a 161-kDa glycogen-binding (G) subunit, the latter being phosphorylated by cAMP-dependent protein kinase at two serine residues (site 1 and site 2). Here the amino acid sequence surrounding site 2 has been determined and this phosphoserine shown to lie 19 residues C-terminal to site 1 in the primary structure. The sequence in this region is: (sequence; see text) At physiological ionic strength, phosphorylation of glycogen-bound PP-1G was found to release all the phosphatase activity from glycogen. The released activity was free C subunit, and not PP-1G, while the phospho-G subunit remained bound to glycogen. Dissociation reflected a greater than or equal to 4000-fold decrease in affinity of C subunit for G subunit and was readily reversed by dephosphorylation. Phosphorylation and dephosphorylation of site 2 was rate-limiting for dissociation and reassociation of C subunit. Release of C subunit was also induced by the binding of anti-site-1 Fab fragments to glycogen-bound PP-1G. At near physiological ionic strength, PP-1G and glycogen concentration, site 2 was autodephosphorylated by PP-1G with a t0.5 of 2.6 min at 30 degrees C, approximately 100-fold slower than the t0.5 for dephosphorylation of glycogen phosphorylase under the same conditions. Site 2 was a good substrate for all three type-2 phosphatases (2A, 2B and 2C) with t0.5 values less than those toward the alpha subunit of phosphorylase kinase. At the levels present in skeletal muscle, the type-2A and type-2B phosphatases are potentially capable of dephosphorylating site 2 in vivo within seconds. Site 1 was at least 10-fold less effective than site 2 as a substrate for all four phosphatases. In conjunction with information presented in the following paper in this issue of this journal, the results substantiate the hypothesis that PP-1 activity towards the glycogen-metabolising enzymes is regulated in vivo by reversible phosphorylation of a targetting subunit (G) that directs the C subunit to glycogen--protein particles. The efficient dephosphorylation of site 2 by the Ca2+/calmodulin-stimulated protein phosphatase (2B) provides a potential mechanism for regulating PP-1 activity in response to Ca2+, and represents an example of a protein phosphatase cascade.     

Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases types 1 and 2A

Cantharidin, a natural toxicant of blister beetles, is a strong inhibitor of protein phosphatases types 1(PP1) and 2A (PP2A). Like okadaic acid, cantharidin inhibits the activity of the purified catalytic subunit of PP2A (IC₅₀ = 0.16 μM) at a lower concentration than that of PPI (IC₅₀ = 1.7 μM) and only inhibits the activity of protein phosphatase type 2B (PP2B) at high concentrations. Dose-inhibition studies conducted with whole cell homogenates indicate that cantharidin also inhibits the native forms of these enzymes. Thus, cantharidin, which is economical and readily available, may be useful as an additional probe for studying the functions of serine/threonine protein phosphatases.     

Activation of brain protein phosphatase-1(I) following cardiac arrest and resuscitation involving an interaction with 14-3-3 gamma

The intracellular signaling mechanisms that couple transient cerebral ischemia to cell death and neuroprotective mechanisms provide potential therapeutic targets for cardiac arrest. Protein phosphatase (PP)-1 is a major serine/threonine phosphatase that interacts with and dephosphorylates critical regulators of energy metabolism, ionic balance, and apoptosis. We report here that PP-1_I, a major regulated form of PP-1, is activated in brain by approximately twofold in vivo following cardiac arrest and resuscitation in a clinically relevant pig model of transient global cerebral ischemia and reperfusion. PP-1_I purified to near homogeneity from either control or ischemic pig brain consisted of the PP-1 catalytic subunit, the inhibitor-2 regulatory subunit, as well as the novel constituents 14-3-3γ, Rab GDP dissociation protein β, PFTAIRE kinase, and C-TAK1 kinase. PP-1_I purified from ischemic brain contained significantly less 14-3-3γ than PP-1_I purified from control brain, and purified 14-3-3γ directly inhibited the catalytic subunit of PP-1 and reconstituted PP-1_I. These findings suggest that activation of brain PP-1_I following global cerebral ischemia in vivo involves dissociation of 14-3-3γ, a novel inhibitory modulator of PP-1_I. This identifies modulation of PP-1_I by 14-3-3 in global cerebral ischemia as a potential signaling mechanism-based approach to neuroprotection.     

Studies of small molecule interactions with protein phosphatases using biosensor technology

Reversible protein phosphorylation of serine, threonine, and tyrosine residues by protein kinases and phosphatases is important for the regulation of cellular signal transduction and controls many cellular functions. Disturbances in this regulation have been implicated in a growing number of diseases, making kinases and phosphatases useful targets for therapeutic intervention. The suitability of surface plasmon resonance (SPR) technology has been widely demonstrated in many drug discovery applications. A novel and straightforward methodology is presented for analyzing small molecule binding to two serine/threonine phosphatases, PP1 and PP2B (calcineurin), and to the prototypic tyrosine phosphatase, PTP1B. Emphasis was placed on investigating the immobilization conditions of the phosphatases by using reducing conditions, inhibitors and metal ions. A comparison of inhibitor binding, either to phosphatase (PP2B) alone or in complex with the regulatory protein subunit calmodulin, revealed different kinetics. The methodology was also used to test inhibitor specificity toward different phosphatases. Inhibition of regulatory protein PP-inhibitor-2 binding to PP1 by a small molecule inhibitor was demonstrated. This type of information, together with data on compound binding that is independent of enzyme activity and in which affinities are resolved into kinetic rate constants, may be of great significance for the development of highly specific and high-affinity phosphatase inhibitors.     

Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A.

Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.     

Protein kinase and phosphatase responses to anoxia in crayfish, Orconectes virilis: purification and characterization of cAMP-dependent protein kinase

The freshwater crayfish, Orconectes virilis, shows good anoxia tolerance, enduring 20 h in N₂-bubbled water at 15°C. Metabolic responses to anoxia by tolerant species often include reversible phosphorylation control over selected enzymes. To analyze the role of serine/threonine kinases and phosphatases in signal transduction during anoxia in O. virilis, changes in the activities of cAMP-dependent protein kinase (PKA) and protein phosphatases 1, 2A, and 2C were measured in tail muscle and hepatopancreas over a time course of exposure to N₂-bubbled water. A strong increase in the percentage of PKA present as the free catalytic subunit (% PKAc) occurred between 1 and 2 h of anoxia exposure whereas phosphatase activities were strongly reduced. This suggests that PKA-mediated events are important in the initial response by tissues to declining oxygen availability. As oxygen deprivation became severe and prolonged (5–20 h) these changes reversed; the % PKAc fell to below control values and activities of phosphatases returned to or rose above control values. Subcellular fractionation also showed a decrease in PKA associated with the plasma membrane after 20 h anoxia whereas cytosolic PKA content increased. PKAc purified from tail muscle showed a molecular weight of 43.8±0.4 kDa, a pH optimum of 6.8, a high affinity for Mg ATP (K_m=131.0±14.4 μM) and Kemptide (K_m=31.6±5.2 μM). Crayfish PKAc was sensitive to temperature change; a break in the Arrhenius plot occurred at approximately 15°C with a 2.5-fold rise in activation energy at temperatures <15°C. These studies demonstrate a role for serine/threonine protein kinases and phosphatases in the metabolic adjustments to oxygen depletion by crayfish organs.