Inhibitor-2 Research Articles

Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1.

The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1-171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.

Is myosin phosphatase regulated in vivo by inhibitor-1? Evidence from inhibitor-1 knockout mice.

1. The Ca(2+) sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor-1 (I-1) is the classic type-1 phosphatase inhibitor, but its presence and role in cAMP-dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I-1 in vivo, we investigated smooth muscle function in a mouse model lacking the I-1 protein (I-1((-/-)) mice). 2. Significant amounts of I-1 protein were detected in the wild-type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by (32)P-labelled aortic extracts from WT mice. 3. Despite the significant presence of I-1 in WT aorta, phenylephrine and KCl concentration- isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I-1((-/-)) aorta. cGMP-dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC(20)), measured in aortic extracts, were nearly identical between WT and I-1((-/-)) mice. 4. In the portal vein, I-1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC(50) of isoproterenol relaxation (EC(50) = 10.4 +/- 1.4 nM) compared to the WT value (EC(50) = 3.5 +/- 0.2 nM). Contraction in response to acetylcholine as well as Ca(2+) sensitivity were similar between WT and I-1((-/-)) aorta. 5. Despite the prevalence of I-1 and its activation by PKA in the aorta, I-1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I-1 may play a role as a fine-tuning mechanism involved in regulating portal vein responsiveness to beta-adrenergic agonists.

Neuronal Cdc2-like Protein Kinase (Cdk5/p25) Is Associated with Protein Phosphatase 1 and Phosphorylates Inhibitor-2

Protein phosphatase 1 (PP1) is complexed with inhibitor 2 (I-2) in the cytosol. In rabbit muscle extract PP1.I-2 is activated upon preincubation with ATP/Mg. This activation is caused by phosphorylation of I-2 on Thr(72) by glycogen synthase kinase 3 (GSK3). We have found that PP1.I-2 in bovine brain extract is also activated upon preincubation with ATP/Mg. However, blocking GSK3 action by LiCl inhibited only approximately 29% of PP1 activity and indicated that GSK3 is not the sole PP1.I-2 activator in the brain. When bovine brain extract was analyzed by gel filtration PP1.I-2 and neuronal Cdc2-like protein kinase (NCLK), a heterodimer of Cdk5 and the regulatory p25 subunit, co-eluted as a approximately 450-kDa size species. The NCLK from the eluted column fractions bound to PP1-specific microcystin-Sepharose and glutathione S-transferase (GST)-I-2-coated glutathione-agarose beads. Similarly, PP1 from the eluted column fractions was pulled down with GST-Cdk5-coated glutathione-agarose beads. In vitro, NCLK phosphorylated I-2 on Thr(72) and activated PP1.I-2 in an ATP/Mg-dependent manner. NCLK bound to PP1 through its Cdk5 subunit and the PP1 binding region was localized to Cdk5 residues 28-41. Our data demonstrate that in brain extract PP1.I-2 and NCLK are associated within a complex of approximately 450 kDa and suggest that NCLK is one of the PP1.I-2-activating kinases in the mammalian brain.

Characterization of a novel serine/threonine protein phosphatase (PfPPJ) from the malaria parasite , Plasmodium falciparum

A novel protein phosphatase cDNA of the PPP superfamily was identified from the malaria parasite, Plasmodium falciparum (Pf), and tentatively named PfPPJ. The predicted primary structure of the phosphatase contained all the known conserved motifs of the PPP superfamily essential for catalytic activity. The enzyme was specific for dephosphorylation of phosphoserine and phosphothreonine residues with very little activity against phosphotyrosine residues. However, the sequence at its C-terminal end was unique, and was consistent with its resistance to the classical PP2A-specific inhibitors such as okadaic acid and microcystin-LR, and the PP1-specific inhibitor, mammalian heat-stable inhibitor-2 (I-2). Even the catalytic core of PfPPJ had a sequence substantially different from the other PPPs such that PfPPJ could be placed in an apparently separate phylogenetic branch. At 294 amino acids residues, PfPPJ was one of the smallest okadaic acid-resistant PPP phosphatases known. By Northern blot analysis, the expression of the PfPPJ mRNA showed the following pattern: schizont>ring>trophozoite, which closely paralleled the expression of the protein, as determined by immunofluorescence. Together, these results suggested a parasitic stage-specific transcriptional regulation of this novel and potentially unique protozoan phosphatase.

A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site

We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 Å) at the active site. This protease-inhibitor binding paradigm is observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising OSer195γ, a water molecule co-bound in the oxyanion hole (H2Ooxy), and the phenolate oxygen atom of the inhibitor (O6′). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin.Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide range of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures suggest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimidazole analogs in which the pKa of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the inhibitor and the enzyme. These data further suggest that the pKa of His57 of each protease in the unbound state in solution is about the same, ∼ 6.8. By comparing inhibition constants (Ki values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity (∼1.7 kcal/mol). The structures and Ki values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn2+-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn2+ complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design.

Interaction of Inhibitor-2 with the Catalytic Subunit of Type 1 Protein Phosphatase: IDENTIFICATION OF A SEQUENCE ANALOGOUS TO THE CONSENSUS TYPE 1 PROTEIN PHOSPHATASE-BINDING MOTIF

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

Cellular Mechanisms Regulating Protein Phosphatase-1: A KEY FUNCTIONAL INTERACTION BETWEEN INHIBITOR-2 AND THE TYPE 1 PROTEIN PHOSPHATASE CATALYTIC SUBUNIT

Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.

Protein Phosphatase-1 Regulation in the Induction of Long-Term Potentiation: Heterogeneous Molecular Mechanisms

Protein phosphatase inhibitor-1 (I-1) has been proposed as a regulatory element in the signal transduction cascade that couples postsynaptic calcium influx to long-term changes in synaptic strength. We have evaluated this model using mice lacking I-1. Recordings made in slices prepared from mutant animals and also in anesthetized mutant animals indicated that long-term potentiation (LTP) is deficient at perforant path-dentate granule cell synapses. In vitro, this deficit was restricted to synapses of the lateral perforant path. LTP at Schaffer collateral-CA1 pyramidal cell synapses remained normal. Thus, protein phosphatase-1-mediated regulation of NMDA receptor-dependent synaptic plasticity involves heterogeneous molecular mechanisms, in both different dendritic subregions and different neuronal subtypes. Examination of the performance of I-1 mutants in spatial learning tests indicated that intact LTP at lateral perforant path-granule cell synapses is either redundant or is not involved in this form of learning.

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.

Drosophila melanogaster protein phosphatase inhibitor-2: identification of a site important for PP1 inhibition

A database search with human protein phosphatase inhibitor-2 (I-2) identified a Drosophila melanogaster cDNA that encoded a protein identical in length and sharing 39% amino acid identity (58% similarity) with human I-2. The mRNA encoding this protein is expressed in both sexes and throughout development, unlike Drosophila inhibitor-t. The bacterially expressed protein was a specific inhibitor of protein phosphatase 1 with an IC 50 of <1 nM, confirming that it is the Drosophila homologue of mammalian inhibitor-2. Mutation of Phe residues conserved in I-2 from lower and higher eukaryotes showed that Phe-33 is important for inhibition of PP1c.

Importance of the β12-β13 Loop in Protein Phosphatase-1 Catalytic Subunit for Inhibition by Toxins and Mammalian Protein Inhibitors

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.

Optimal sequences for non-phosphate-directed phosphorylation by protein kinase CK1 (casein kinase-1)--a re-evaluation.

A variety of synthetic peptides derived from either the inhibitor-2 (I-2) phosphoacceptor sites or the optimal sequences selected in an oriented peptide library have been compared for their susceptibility to phosphorylation by protein kinase CK1 (also termed casein kinase-1). The I-2-derived peptides are by far preferred over the library peptides by both rat liver CK1 (and by the alpha/beta, gamma and delta/epsilon isoforms immunoprecipitated from it) and recombinant Xenopus laevis CK1 alpha. The superiority of the I-2-derived peptides over the library ones is reflected by Vmax values one to two orders of magnitude higher while the Km values are comparable. Individual substitutions of any of the aspartic acids with alanine in the I-2-derived peptide RRKHAAIGDDDDAYSITA is detrimental, producing both a fall in Vmax and an increase in Km which are more pronounced at position n -3, but also quite significant at positions n -4, n -5 and, to a lesser extent, n -6. The unfavourable effect of these substitutions is more evident with rat liver CK1 than with recombinant Xenopus laevis CK1 alpha. The chimeric peptide IGDDDDAY-S-IIIFFA, resulting from the combination of the N-terminal acidic sequence of the I-2 (Ser86) site and the C-terminal hydrophobic cluster selected in the library peptides (MAEFDTG-S-IIIFFAKKK and MAYYDAA-S-IIIFFAKKK) is phosphorylated as efficiently as the I-2-derived peptide in terms of both Km and Vmax. These combined data strongly support the conclusion that, at variance with the optimal sequences selected in the library, optimal non-phosphate-directed phosphorylation of peptide substrates by CK1 critically relies on the presence of a cluster of acidic residues (preferably aspartic acid) upstream from position n -2, while the highly hydrophobic region downstream from serine selected in the library appears to be dispensable. The reason for these discrepancies remains unclear. The possibility that the library data are biased by the invariant elements forming its scaffold (MA-x-x-x-x-x-SI-x-x-x-x-AKKK) would be consistent with the observation that the library-selected peptides, despite their low Km values, fail to compete against the phosphorylation of protein and peptide substrates by CK1, suggesting that they bind to elements partially distinct from those responsible for substrate recognition.

A Model Phosphatase 2C → Phosphatase 1 Activation Cascade via Dual Control of Inhibitor-1 (INH-1) and DARPP-32 Dephosphorylation by Two Inositol Glycan Putative Insulin Mediators from Beef Liver

Two inositol phosphoglycans (IPG) isolated from beef liver and designated as putative insulin mediators were demonstrated to reciprocally enhance the dephosphorylation of inhibitor-1 (INH-1) and DARPP-32, thus directly activating phosphatase 2C and disinhibiting phosphatase 1 in a potential protein phosphatase 2C → phosphatase 1 cascade mechanism. One IPG termed pH 2.0, containing Dchiro-inositol and galactosamine, stimulated the dephosphorylation of INH-1 and DARPP-32 in a dose-dependent manner in the low micromolar range. A second, termed pH 1.3, containing myo-inositol glucosamine and mannose acted reciprocally to inhibit the cAMP-dependent protein kinase phosphorylation of INH-1 and DARPP-32 in a dose-dependent manner in the low micromolar range. These model experiments are discussed in terms of the observed dephosphorylation of INH-1 with insulin action documented in the literature and the activation of both phosphatase 1 and 2C described in intact cells andin vivowith insulin action.

Characterization of protein serine/threonine phosphatase activities in human lung mast cells and basophils.

1. The serine/threonine protein phosphatase (PP) inhibitors, okadaic acid and calyculin, attenuated the IgE-mediated release of histamine from human lung mast cells (HLMC) and basophils in a dose-dependent manner whereas an alternative PP inhibitor, microcystin, was ineffective. Calyculin was more potent than okadaic acid in both cell types. The concentration required to inhibit by 50% (IC50) the release of histamine was 15 (HLMC) and 50 nM (basophils) for calyculin and 200 (HLMC) and 300 nM (basophils) for okadaic acid. 2. Lysates of purified HLMC and basophils dephosphorylated radiolabelled glycogen phosphorylase, a substrate for both PP1 and PP2A. The PP activity in lysates of both cell types was inhibited in a dose-dependent fashion by the PP inhibitors with the following rank order of activity, calyculin (approximate IC50; 0.02-0.1 nM) > or = microcystin (0.1 nM) > okadaic acid (70 nM). 3. The PP1-selective inhibitor, inhibitor-2 (I-2), attenuated the dephosphorylation of glycogen phosphorylase in lysates of both HLMC and basophils. I-2 (20 nM) inhibited the glycogen phosphorylase PP activity by 71+/-3% and 49+/-13% in HLMC and basophil extracts, respectively. There were, approximately, 6 fold greater levels of I-2-sensitive activity in HLMC than in basophils. Qualitatively similar results were obtained with an alternative PP1-selective inhibitor, inhibitor-1 (I-1). 4. Lysates derived from HLMC and basophils dephosphorylated radiolabelled casein which is a PP2A-restricted substrate. HLMC lysates contained, approximately, 2.5 fold higher levels of casein PP activity than basophil lysates. 5. These data indicate that HLMC and basophils both contain PP1 and PP2A. The data suggest that, on a per cell basis, HLMC have higher levels of both PP1 and PP2A. Moreover, the ratio of PP1 to PP2A is higher in HLMC than in basophils.

Inhibitor-1 Interaction Domain That Mediates the Inhibition of Protein Phosphatase-1

Inhibitor-1 (I-1), a cyclic AMP-regulated phosphoprotein, inhibits protein phosphatase-1 (PP1) activity in response to hormones. The molecular mechanism for PP1 inhibition by I-1 remains unknown. Mutation of nine acidic residues lining a proposed I-1-binding channel in rabbit PP1alpha yielded one mutant (E256A) slightly impaired in its inhibition by I-1, with the IC50 increased by 3-fold, and one mutant (E275R) located in the beta12-beta13 loop that showed 4-fold enhanced inhibition by I-1. Substituting Tyr-272, a proposed binding site for the toxins okadaic acid and microcystin-LR, in the beta12-beta13 loop with Trp, Phe, Asp, Arg, or Ala impaired PP1alpha inhibition by I-1 by 8-10-fold. Chemical mutagenesis of the Saccharomyces cerevisiae PP1 gene (GLC7) yielded 20 point mutations in the PP1 coding region. Two-hybrid analyses and biochemical assays of these yeast enzymes identified four additional residues in the beta12-beta13 loop that were required for PP1 binding and inhibition by I-1. Ten-fold higher concentrations of I-1 were required to inhibit these mutants. Finally, deletion of the beta12-beta13 loop from PP1alpha maintained full enzyme activity, but attenuated inhibition by I-1 by >100-fold. These data identified the beta12-beta13 loop in the PP1 catalytic subunit as a domain that mediates binding and enzyme inhibition by I-1.

Intracellular effects of okadaic acid in the blue mussel Mytilus edulis, and rainbow trout Oncorhynchus mykiss

The effects of okadaic acid (OA) on protein phosphatase (PP) and glycogen synthase (GS) activity was investigated in the blue mussel and rainbow trout. In both species, PP activity was inhibited by 200 nM inhibitor-2 (I-2) as well as 2 nM OA indicating the presence of both type 1 and type 2A phosphatases. GS activity was measured in an in vitro tissue slice method. Ten μM OA inhibited GS activity in rainbow trout but did not alter GS activity in the blue mussel. Also, feeding the mussels the OA-producing algae Prorocentrum lima had no effect on GS. These results seem to indicate that there may be protective mechanisms against harmful effects of OA in the blue mussel.

Cloning and in vitro expression of TPK3, a Toxoplasma gondii homologue of shaggy/glycogen synthase kinase-3 kinases

As an initial effort to dissect the signaling pathways responsible for pathogenesis of Toxoplasma gondii infection, we report the cloning and in vitro functional studies of TPK3 ( Toxoplasma protein kinase-3), a homologue of shaggy/glycogen synthase kinase-3 (GSK-3) kinases. The shaggy/GSK-3 family of kinases are highly conserved protein kinases that play important roles in cell fate determination, nuclear signaling and hormonal regulation. The TPK3 gene was isolated by RT-PCR with degenerate primers corresponding to highly conserved regions of serine/threonine protein kinases. The complete sequences of genomic and cDNA clones indicated the open reading frame, 1185 bp in size, is interrupted by five introns. The predicted protein sequence of TPK3 shows 54% identity to shaggy/GSK-3 over the catalytic domains. Southern analysis revealed TPK3 is a single copy locus in the Toxoplasma genome. Antisera to other GSK-3 proteins from other species recognized GST-TPK3 and a protein of the predicted size in parasites lysates. In vitro kinase assays with GST-TPK3 indicated that TPK3 autophosphorylates and phosphorylates protein phosphatase inhibitor-2 (I-2), a specific substrate of GSK-3 kinase.

Relationships of subunits of type-1 serine/threonine protein phosphatase to morphology and aggregation of B cells.

To elucidate the roles of serine/threonine protein phosphatases PP1 and PP2A in the morphological changes of B-lymphocytes during development and in immune responses, we investigated alterations of protein levels of catalytic subunits of PP1 and PP2A and regulatory subunits of PP1 including M130/M133, inhibitor-1 (I-1) and inhibitor-2 (I-2) in B-cell lines at different maturational stages and during their aggregation induced by phorbol myristate acetate (PMA). The protein levels of PP1delta and/or M130/M133 were significantly lower in B-cell lines without pseudopods, WEHI-231, BAL-17, Daudi, and CESS, than in those with pseudopods, Bcl.1, A20, M12, and SKW6.4, whereas the amounts of PP1alpha and PP2A were similar among them. During aggregation of A20 and CESS cells induced by PMA, an activator of PKC, the amount of PP1delta was progressively decreased, and this decrease was blocked by H7, an inhibitor of PKC. The amount of PP1alpha was constant under these conditions. Okadaic acid, an inhibitor of PP1 and PP2A, also induced aggregation of A20 cells at concentrations sufficient to inhibit PP1, but not at lower concentrations that inhibit PP2A alone. These results suggest that myosin light chain phosphatase composed of PP1delta and M130/M133 is involved in the maintenance and regulation of cytoskeletal structures in B-lymphocytes.

Genetic Analysis of Human Type 1 Protein Phosphatase Inhibitor 2 in Insulin-Resistant Pima Indians

The rate-limiting enzyme in insulin-mediated nonoxidative glucose disposal, glycogen synthase, has reduced activity in insulin-resistant subjects at risk for developing non-insulin-dependent diabetes mellitus (NIDDM). The synthase-activating enzyme, type 1 protein phosphatase (PP1), also has an abnormally low level of activity. Inhibitor 2 (I-2) reversibly inhibits and facilitates the proper conformation of free catalytic subunits of PP1. This study investigates whether genetic alteration(s) in the I-2 coding locus (PPP1R2) could contribute to insulin resistance in Pima Indians. We determined that the authenticPPP1R2gene is located on chromosome 3q29 and consists of six exons. The previously reported homologue ofPPP1R2on chromosome 5 is identified as an intronless pseudogene. Comparative sequencing ofPPP1R2exons and splice junctions revealed no mutations in insulin-resistant Pima Indians. The information on the genomic structure ofPPP1R2is important for exploring this gene as a potential candidate contributing to insulin resistance and NIDDM in other populations.

Identification and Characterization of three Isotypes of Protein Phosphatase Inhibitor‐2 and their Expression Profiles during Testis Maturation in Rats

cDNAs for three isotypes of inhibitor-2(I-2), I-2 alpha 1, I-2 alpha 2 and I-2 beta were isolated from a rat testis library. I-2 alpha 2 and I-2 beta are new forms. The former is an alternatively spliced form of I-2 alpha 1, encoding a protein with substitution of three amino acids for 14 amino acids of the I-2 alpha 1 protein at the C-terminus. The latter is derived from a different gene and encodes a 126-amino-acid protein having highly conserved regions with the I-2 alpha 1 protein from amino acid positions 22-47 and 111-126. I-2 alpha 2 and I-2 beta are expressed exclusively in the testis, and the expressions of all three forms of I-2 coincide with sperm cell maturation. The half-maximal inhibitory concentration of the GST-I-2 alpha 2 fusion protein on the PP1 gamma 2 catalytic subunit is the same as that of GST-I-2 alpha 1, being 10 nM. However, the half-maximal inhibitory concentration of GST-1-2 beta is 100-fold higher, being 1 microM. GST-I-2 beta showed no competition with GST-I-2 alpha 1. and its biological significance is unknown.