Flexible Tethering of ASPP Proteins Facilitates PP-1c Catalysis.

ASPP (apoptosis-stimulating proteins of p53) proteins bind PP-1c (protein phosphatase 1) and regulate p53 impacting cancer cell growth and apoptosis. Here we determine the crystal structure of the oncogenic ASPP protein, iASPP, bound to PP-1c. The structure reveals a 1:1 complex that relies on interactions of the iASPP SILK and RVxF motifs with PP-1c, plus interactions of the PP-1c PxxPxR motif with the iASPP SH3 domain. Small-angle X-ray scattering analyses suggest that the crystal structure undergoes slow interconversion with more extended conformations in solution. We show that iASPP, and the tumor suppressor ASPP2, enhance the catalytic activity of PP-1c against the small-molecule substrate, pNPP as well as p53. The combined results suggest that PxxPxR binding to iASPP SH3 domain is critical for complex formation, and that the modular ASPP-PP-1c interface provides dynamic flexibility that enables functional binding and dephosphorylation of p53 and other diverse protein substrates.

TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cß.

Transforming growth factor-ß membrane associated protein (TIMAP) is an endothelial cell (EC)-predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein phosphatase 1 subunit PP1cß, forming myosin phosphatase holoenzymes. We investigated whether TIMAP/PP1cß could also function as a myosin phosphatase. Endogenous PP1cß, myosin light chain 2 (MLC2), and myosin IIA heavy chain coimmunoprecipitated from EC lysates with endogenous TIMAP, and endogenous MLC2 colocalized with TIMAP in EC projections. Purified recombinant GST-TIMAP interacted directly with purified recombinant His-MLC2. However, TIMAP overexpression in EC enhanced MLC2 phosphorylation, an effect not observed with a TIMAP mutant that does not bind PP1cß. Conversely, MLC2 phosphorylation was reduced in lung lysates from TIMAP-deficient mice and upon silencing of endogenous TIMAP expression in ECs. Ectopically expressed TIMAP slowed the rate of MLC2 dephosphorylation, an effect requiring TIMAP-PP1cß interaction. The association of MYPT1 with PP1cß was profoundly reduced in the presence of excess TIMAP, leading to proteasomal MYPT1 degradation. In the absence of TIMAP, MYPT1-associated PP1cß readily bound immobilized microcystin-LR, an active-site inhibitor of PP1c. By contrast, TIMAP-associated PP1cß did not interact with microcystin-LR, indicating that the active site of PP1cß is blocked when it is bound to TIMAP. Thus, TIMAP inhibits myosin phosphatase activity in ECs by competing with MYPT1 for PP1cß and blocking the PP1cß active site.

Conserved residues in the N terminus of lipin-1 are required for binding to protein phosphatase-1c, nuclear translocation, and phosphatidate phosphatase activity.

Lipin-1 is a phosphatidate phosphatase in glycerolipid biosynthesis and signal transduction. It also serves as a transcriptional co-regulator to control lipid metabolism and adipogenesis. These functions are controlled partly by its subcellular distribution. Hyperphosphorylated lipin-1 remains sequestered in the cytosol, whereas hypophosphorylated lipin-1 translocates to the endoplasmic reticulum and nucleus. The serine/threonine protein phosphatase-1 catalytic subunit (PP-1c) is a major protein dephosphorylation enzyme. Its activity is controlled by interactions with different regulatory proteins, many of which contain conserved RVXF binding motifs. We found that lipin-1 binds to PP-1cγ through a similar HVRF binding motif. This interaction depends on Mg(2+) or Mn(2+) and is competitively inhibited by (R/H)VXF-containing peptides. Mutating the HVRF motif in the highly conserved N terminus of lipin-1 greatly decreases PP-1cγ interaction. Moreover, mutations of other residues in the N terminus of lipin-1 also modulate PP-1cγ binding. PP-1cγ binds poorly to a phosphomimetic mutant of lipin-1 and binds well to the non-phosphorylatable lipin-1 mutant. This indicates that lipin-1 is dephosphorylated before PP-1cγ binds to its HVRF motif. Importantly, mutating the HVRF motif also abrogates the nuclear translocation and phosphatidate phosphatase activity of lipin-1. In conclusion, we provide novel evidence of the importance of the lipin-1 N-terminal domain for its catalytic activity, nuclear localization, and binding to PP-1cγ.

Multi-directional function of the protein phosphatase 1 regulatory subunit TIMAP.

TIMAP is an endothelial-cell predominant member of the MYPT family of PP1c regulatory subunits. This study explored the TIMAP-PP1c interaction and substrate specificity in vitro. TIMAP associated with all three PP1c isoforms, but endogenous endothelial cell TIMAP preferentially co-immunoprecipitated with PP1cß. Structural modeling of the TIMAP/PP1c complex predicts that the PP1c C-terminus is buried in the TIMAP ankyrin cluster, and that the PP1c active site remains accessible. Consistent with this model, C-terminal PP1c phosphorylation by cdk2-cyclinA was masked by TIMAP, and PP1c bound TIMAP when the active site was occupied by the inhibitor microcystin. TIMAP inhibited PP1c activity toward phosphorylase a in a concentration-dependent manner, with half-maximal inhibition in the 0.4-1.2 nM range, an effect modulated by the length, and by Ser333/Ser337 phosphomimic mutations of the TIMAP C-terminus. TIMAP-bound PP1cß effectively dephosphorylated MLC2 and TIMAP itself. By contrast, TIMAP inhibited the PP1cß activity toward the putative substrate LAMR1, and instead masked LAMR1 PKA- and PKC-phosphorylation sites. This is direct evidence that MLC2 is a TIMAP/PP1c substrate. The data also indicate that TIMAP can modify protein phosphorylation independent of its function as a PP1c regulatory subunit, namely by masking phosphorylation sites of binding partners like PP1c and LAMR1.

Molecular mechanisms underlying the interaction of protein phosphatase-1c with ASPP proteins.

The serine/threonine PP-1c (protein phosphatase-1 catalytic subunit) is regulated by association with multiple regulatory subunits. Human ASPPs (apoptosis-stimulating proteins of p53) comprise three family members: ASPP1, ASPP2 and iASPP (inhibitory ASPP), which is uniquely overexpressed in many cancers. While ASPP2 and iASPP are known to bind PP-1c, we now identify novel and distinct molecular interactions that allow all three ASPPs to bind differentially to PP-1c isoforms and p53. iASPP lacks a PP-1c-binding RVXF motif; however, we show it interacts with PP-1c via a RARL sequence with a Kd value of 26 nM. Molecular modelling and mutagenesis of PP-1c-ASPP protein complexes identified two additional modes of interaction. First, two positively charged residues, Lys260 and Arg261 on PP-1c, interact with all ASPP family members. Secondly, the C-terminus of the PP-1c α, ß and γ isoforms contain a type-2 SH3 (Src homology 3) poly-proline motif (PxxPxR), which binds directly to the SH3 domains of ASPP1, ASPP2 and iASPP. In PP-1cγ this comprises residues 309-314 (PVTPPR). When the Px(T)PxR motif is deleted or mutated via insertion of a phosphorylation site mimic (T311D), PP-1c fails to bind to all three ASPP proteins. Overall, we provide the first direct evidence for PP-1c binding via its C-terminus to an SH3 protein domain.

Lethal, hereditary mutants of phospholamban elude phosphorylation by protein kinase A.

The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by ß-adrenergic stimulation and phosphorylation of phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.

Identification of a small molecule inhibitor of the human DNA repair enzyme polynucleotide kinase/phosphatase.

Human polynucleotide kinase/phosphatase (hPNKP) is a 57.1-kDa enzyme that phosphorylates DNA 5'-termini and dephosphorylates DNA 3'-termini. hPNKP is involved in both single- and double-strand break repair, and cells depleted of hPNKP show a marked sensitivity to ionizing radiation. Therefore, small molecule inhibitors of hPNKP should potentially increase the sensitivity of human tumors to gamma-radiation. To identify small molecule inhibitors of hPNKP, we modified a novel fluorescence-based assay to measure the phosphatase activity of the protein, and screened a diverse library of over 200 polysubstituted piperidines. We identified five compounds that significantly inhibited hPNKP phosphatase activity. Further analysis revealed that one of these compounds, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), was the most effective, with an IC50 of 0.06 micromol/L. When tested for its specificity, A12B4C3 displayed no inhibition of two well-known eukaryotic protein phosphatases, calcineurin and protein phosphatase-1, or APTX, another human DNA 3'-phosphatase, and only limited inhibition of the related PNKP from Schizosaccharomyces pombe. At a nontoxic dose (1 micromol/L), A12B4C3 enhanced the radiosensitivity of human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells by a factor of two, which was almost identical to the increased sensitivity resulting from shRNA-mediated depletion of hPNKP. Importantly, A12B4C3 failed to increase the radiosensitivity of the hPNKP-depleted cells, implicating hPNKP as the principal cellular target of A12B4C3 responsible for increasing the response to radiation. A12B4C3 is thus a useful reagent for probing hPNKP cellular function and will serve as the lead compound for further development of PNKP-targeting drugs.

Protein phosphatase inhibitors isolated from Spongia irregularis collected in Papua New Guinea.

Irregularasulfate (1), a new nitrogen-containing sesterterpenoid, and the known sesterterpenoids hipposulfate C (2), halisulfate-7 (3), and igernellin (4), have been isolated from the marine sponge Spongia irregularis collected in Papua New Guinea. The structure of 1 was elucidated via analysis of its spectroscopic data. Sesterterpenoids 1, 2, and 3 are moderate inhibitors of the catalytic subunits of the mammalian Ser/Thr protein phosphatases calcineurin, PP-1, and PP-2A. The phosphate analogue of 3 and the thiophosphate analogue of 2 have been prepared from the corresponding natural products and evaluated for their ability to inhibit the phosphatase activity of calcineurin.

Tissue distribution and oral dose effects of microcystin in the freshwater pulmonate snail Lymnaea stagnalis jugularis (Say).

Microcystin (MC) concentrations were measured in the alimentary tract, digestive gland, and remaining visceral mass of adult pulmonate snails (Lymnaea stagnalis) exposed to cyanobacteria known to contain MC. The highest proportion of total body MC content was measured within the alimentary tract (83%), though an appreciable proportion (17%) was also found within the digestive gland tissue. This provides conclusive evidence for the limited digestion of toxic cyanobacteria and subsequent uptake and accumulation of MC by the digestive gland of L. stagnalis. Additionally, pure microcystin-LR was orally administered to adult L. stagnalis to investigate the potential for toxic effects. Exposure to microcystin-LR induced histopathological alterations of the digestive glands consistent with those reported elsewhere for mammals and fish, indicating a common mode of toxicity to both vertebrates and invertebrates.

Elimination of the cyanobacterial hepatotoxin microcystin from the freshwater pulmonate snail Lymnaea stagnalis jugularis (say).

It has been suggested that little to no microcystin (MC), a cyanobacterial hepatotoxin, accumulates within freshwater pulmonate snails because the toxin is associated primarily with undigested gut contents that are eliminated from the animal via egestion. To test this, Lymnaea stagnalis exposed to MC-containing cyanobacteria were placed into toxin-free environments and sampled over short (24 h at 21 degrees C) and long (30 d at 22 and 10 degrees C) time periods. Within 8 h after being removed from exposure to microcystin-containing phytoplankton, the gizzard and cecal string fractions of the feces were eliminated, accounting for 57% of the initial MC concentration. However, detectable concentrations remained beyond 24 h, likely in association with the digestive-gland contents, which can be retained up to 100 h. Long-term MC loss was biphasic at two ambient temperatures. The greatest change (fast phase) occurred over the first 3 d after exposure. By 6 d, the cumulative MC loss from L. stagnalis was 80 and 95% at 10 and 22 degrees C, respectively. Toxin loss over this period was attributed to egestion of indigestible cells/colonies from gizzard and cecum, as well as elimination of unassimilated MC-laden fragments and vacuolate excretion of residues from the digestive gland. The fast-phase depuration rate constant was significantly higher at 22 than at 10 degrees C, indicating an influence of ambient temperature on the rate of toxin loss from pulmonate snails. Depuration continued at slower rates until 30 d, when most (97.5 and 99.5% at 10 and 22 degrees C, respectively) of the initial MC was eliminated.

Crystal structures of protein phosphatase-1 bound to motuporin and dihydromicrocystin-LA: elucidation of the mechanism of enzyme inhibition by cyanobacterial toxins.

The microcystins and nodularins are tumour promoting hepatotoxins that are responsible for global adverse human health effects and wildlife fatalities in countries where drinking water supplies contain cyanobacteria. The toxins function by inhibiting broad specificity Ser/Thr protein phosphatases in the host cells, thereby disrupting signal transduction pathways. A previous crystal structure of a microcystin bound to the catalytic subunit of protein phosphatase-1 (PP-1c) showed distinct changes in the active site region when compared with protein phosphatase-1 structures bound to other toxins. We have elucidated the crystal structures of the cyanotoxins, motuporin (nodularin-V) and dihydromicrocystin-LA bound to human protein phosphatase-1c (gamma isoform). The atomic structures of these complexes reveal the structural basis for inhibition of protein phosphatases by these toxins. Comparisons of the structures of the cyanobacterial toxin:phosphatase complexes explain the biochemical mechanism by which microcystins but not nodularins permanently modify their protein phosphatase targets by covalent addition to an active site cysteine residue.

Insulin, glucagon and fatty acid treatment of hepatocytes does not result in phosphorylation or changes in activity of triacylglycerol hydrolase.

It is recognized that the majority of very low density lipoprotein (VLDL) associated triacylglycerol (TG) is synthesized from fatty acids and partial acylglycerols generated by lipolysis of intra-hepatic storage rather than made de novo. Triacylglycerol hydrolase (TGH) is involved in mobilizing stored TG. Modulating the ability of TGH to hydrolyze stored lipids represents a potentially regulated and rate limiting step in VLDL assembly. Phosphorylation of lipases and carboxylesterases trigger diverse but functionally significant events. We explored the potential for regulating the mobilization of hepatic TG through phosphorylation of TGH. Insulin is known to suppress VLDL secretion from liver, and glucagon can be considered an opposing hormone. However, neither insulin nor glucagon treatment of hepatocytes led to phosphorylation of TGH or changes in its activity. Augmenting intracellular TG stores by incubations with oleic acid also did not lead to changes in TGH activity. Therefore, changes in phosphorylation state are not a mechanism for regulating TGH activity, access to TG substrate pools or for TGH-mediated contributions to VLDL assembly and secretion.

Protein phosphatase regulation of Na+/H+ exchanger isoform I.

We investigated regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) by dephosphorylation. Treatment of primary cultures of cardiomyocytes with the phosphatase inhibitor okadaic acid increased the rate of recovery from an acid load, suggesting that the okadaic acid sensitive PP1 may be involved in NHE1 regulation in vivo. We examined the ability of purified protein phosphatases PP1, PP2A, and PP2B to dephosphorylate the regulatory cytoplasmic tail. NHE1 was completely dephosphorylated by PP1, poorly dephosphorylated by PP2A, and not dephosphorylated by PP2B. Examination of NHE1 binding to PP1 or PP2B revealed that an association occurs between NHE1 and PP1 both in vitro and in vivo, but NHE1 did not associate with full-length PP2B. We expressed PP1 or inhibitor 2, a specific PP1 inhibitor, in cell lines to examine the effect of PP1 on NHE1 activity in vivo. Overexpression of PP1 causes a decrease in NHE1 activity but does not affect stimulation by thrombin. Cell lines expressing the specific PP1 inhibitor, inhibitor 2, had elevated proton efflux rates and could not be further stimulated by the Na(+)/H(+) exchanger agonist thrombin. The results suggest that PP1 is an important regulatory phosphatase of NHE1, that it can bind to and dephosphorylate the protein, and that it regulates NHE1 activity in vivo.

Activation of serine/threonine protein phosphatase-1 is required for ceramide-induced survival of sympathetic neurons.

In sympathetic neurons, C6-ceramide, as well as endogenous ceramides, blocks apoptosis elicited by NGF (nerve growth factor) deprivation. The mechanism(s) involved in ceramide-induced neuronal survival are poorly understood. Few direct targets for the diverse cellular effects of ceramide have been identified. Amongst those proposed is PP-1c, the catalytic subunit of serine/threonine PP-1 (protein phosphatase-1). Here, we present the first evidence of PP-1c activation by ceramide in live cells, namely NGF-deprived sympathetic neurons. We first determined PP activity in cellular lysates from sympathetic neurons treated with exogenous ceramide and demonstrated a 2-3-fold increase in PP activity. PP activation was completely blocked by the addition of the specific type-1 PP inhibitor protein I-2 as well as by tautomycin, but unaffected by 2 nM okadaic acid, strongly indicating that the ceramide-activated phosphatase activity was PP-1c. Inhibition of PP activity by phosphatidic acid (which has been reported to be a selective inhibitor of PP-1c) and tautomycin (a PP-1 and PP-2A inhibitor), but not by 10 nM okadaic acid, abolished the anti-apoptotic effect of ceramide in NGF-deprived neurons, suggesting that activation of PP-1c is required for ceramide-induced neuronal survival. Ceramide was able to prevent pRb (retinoblastoma gene product) hyperphosphorylation by a mechanism dependent on PP-1c activation, suggesting that two consequences of NGF deprivation in sympathetic neurons are inhibition of PP-1c and subsequent hyperphosphorylation of pRb protein. These findings suggest a novel mechanism for ceramide-induced survival, and implicate the involvement of PPs in apoptosis induced by NGF deprivation.

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.

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication.

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.