Membrane targeting with palmitoylated lysine added to PP1-disrupting peptide induces PP1-independent signaling

Protein phosphatase-1 (PP1) is a ubiquitous enzyme involved in multiple processes inside cells. PP1-disrupting peptides (PDPs) are chemical tools that selectively bind to PP1 and release its activity. To restrict the activity of PDPs to a cellular compartment, we developed PDP-Mem, a cell membrane-targeting PDP. The membrane localization was achieved through the introduction of a palmitoylated lysine. PDP-Mem was shown to activate PP1α in vitro and to localize to the membrane of HeLa Kyoto and U2OS cells. However, in cells, the combination of the polybasic sequence for cell penetration and the membrane targeting palmitoylated lysine activates the MAPK signaling pathway and induces cytoplasmic calcium release independently of PP1 activation. Therefore, when targeting peptides to cellular membranes, undesired effects induced by the targeting sequence and lipid modification need to be considered.     

Towards a comprehensive analysis of the protein phosphatase 1 interactome in Drosophila

Protein phosphatase type 1 (PP1) is one of the major classes of serine/threonine protein phosphatases, and has been found in all eukaryotic cells examined to date. Metazoans from Drosophila to humans have multiple genes encoding catalytic subunits of PP1 (PP1c), which are involved in a wide range of biological processes. Different PP1c isoforms have pleiotropic and overlapping functions; this has complicated the analysis of their biological roles and the identification of specific in vivo substrates. PP1c isoforms are associated in vivo with regulatory subunits that target them to specific locations and modify their substrate specificity and activity. The PP1c-binding proteins are therefore the key to understanding the role of PP1 in particular biological processes. The existence of isoform specific PP1c-binding subunits may also help to explain the unique roles of different PP1c isoforms. Here we report the identification of 24 genes encoding Drosophila PP1c-binding proteins in the yeast two-hybrid system. Sequence analysis identified a minimal interacting fragment and putative PP1c-binding motif for each protein, delimiting the region involved in binding to PP1c. Further two-hybrid analysis showed that virtually all of the interactors were capable of binding all Drosophila PP1c isoforms. One of the novel interactors, CG1553, was examined further and shown to interact with multiple isoforms by co-immunoprecipitation from Drosophila extracts and functional interaction with PP1c isoforms in vivo. Bioinformatic analyses implicate the putative PP1c-associated subunits in a diverse array of intracellular processes. Our identification of a large number of PP1c-binding proteins with the potential for directing PP1c's specific functions in Drosophila represents a significant step towards a full understanding of the range of PP1 complexes and function in animals.     

The metabotropic glutamate receptor mGluR7b binds to the catalytic gamma-subunit of protein phosphatase 1

Correct targeting of enzymes represents an important biological mechanism to control post-translational modifications of neurotransmitter receptors. The metabotropic glutamate receptor type 7 (mGluR7) exists in two splice variants (mGluR7a and mGluR7b), defined by different C-termini that are phosphorylated by protein kinase C (PKC). Recently, the search for mGluR7a binding partners yielded several proteins that interacted with its C-terminus. Here, a yeast two-hybrid screen using the mGluR7b C-terminus identified both variants of the catalytic gamma-subunit of protein phosphatase 1 (PP1gamma1 and PP1gamma2) as binding partners. The minimal interacting region of PP1gamma1/2 contained the core domain and was homologous to a region of PP1alpha that is needed for functional expression. Although this core domain is highly conserved within the protein phosphatase family, PP1alpha1 and PP1beta did not interact with mGluR7b. Binding between PP1gamma1 and mGluR7b might be regulated by alternative splicing, as the variant-specific distal part of the mGluR7b C-terminus mediated the interaction. Within this domain, amino acids involved in the binding to PP1gamma1 were mapped and biochemical assays using recombinant and native proteins verified the proposed interaction. Finally, the expression pattern of PP1gamma1, PP1gamma2 and mGluR7b was analysed in various CNS regions. In summary, these results suggest a regulation of mGluR7b by PP1gamma.     

Identification and domain mapping of Dictyostelium discoideum type-1 protein phosphatase inhibitor-2

The protein phosphatase type-1 catalytic subunit (PP1c) does not exist freely in the cell and its activity must be very strictly controlled. Several protein inhibitors of PP1c have been described including the classical mammalian inhibitor-1 (I-1) and inhibitor-2 (I-2). Association of these inhibitors with PP1c appears to involve multiple contacts and in the case of I-2 no less than five I-2 interaction subdomains have been proposed. In this report, we provide both in vitro and in vivo evidence that the Dictyostelium discoideum genome encodes a protein (DdI-2) that is an ortholog of mammalian I-2, being the first PP1c interacting protein characterized in this social amoeba. Despite the low overall sequence similarity of DdI-2 with other I-2 sequences and its long N-terminal extension, the five PP1c interaction motifs proposed for mammalian I-2 are reasonably conserved in the Dictyostelium ortholog. We demonstrate that DdI-2 interacts with and inhibits D. discoideum PP1c (DdPP1c), which we have previously characterized. Moreover, using yeast two-hybrid assays we show that a stable interaction of DdI-2 with DdPP1c requires multiple contacts.     

Plasmodium berghei leucine-rich repeat protein 1 downregulates protein phosphatase 1 activity and is required for efficient oocyst development

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1–LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.     

Nuclear envelope precursor vesicle targeting to chromatin is stimulated by protein phosphatase 1 in Xenopus egg extracts

The mechanism underlying targeting of the nuclear membrane to chromatin at the end of mitosis was studied using an in vitro cell-free system comprising Xenopus egg membrane and cytosol fractions, and sperm chromatin. The mitotic phase membrane, which was separated from a mitotic phase extract of Xenopus eggs and could not bind to chromatin, became able to bind to chromatin on pretreatment with a synthetic phase cytosol fraction of Xenopus eggs. When the cytosol fraction was depleted of protein phosphatase 1 (PP1) with anti-Xenopus PP1γ1 antibodies, this ability was lost. The addition of recombinant xPP1γ1 to the PP1-depleted cytosol fraction restored the ability. These and other results suggested that dephosphorylation of mitotic phosphorylation sites on membranes by PP1 in the synthetic phase cytosol fraction promoted targeting of the membranes to chromatin. On the other hand, a fragment containing the chromatin-binding domain of lamin B receptor (LBR) but not emerin inhibited targeting of membrane vesicles. It was also shown that PP1 dephosphorylates a phosphate group(s) responsible for regulation of the binding of LBR to chromatin. A possible mechanism involving PP1 and LBR for the regulation of nuclear membrane targeting to chromatin was discussed.     

PAI1: a novel PP1‐interacting protein that mediates human plasma's anti‐apoptotic effect in endothelial cells

Activation of apoptotic signalling in endothelial cells contributes to the detrimental effects of a variety of pathological stimuli. In investigating the molecular events underlying the anti‐apoptotic effect of human plasma in cultured human endothelial cells, we unexpectedly uncovered a novel mechanism of apoptosis suppression by human plasma through an interaction between two previously unrelated proteins. Human plasma inhibited hypoxia–serum deprivation‐induced apoptosis and stimulated BAD^S136 and Akt^S473 phosphorylation. Akt1 silencing reversed part (~52%) of the anti‐apoptotic effect of human plasma, suggesting the existence of additional mechanisms mediating the anti‐apoptotic effect other than Akt signalling. Human plasma disrupted the interaction of BAD with protein phosphatase 1 (PP1). Mass spectrometry identified fourteen PP1‐interacting proteins induced by human plasma. Notably, a group of serine protease inhibitors including plasminogen activator inhibitor 1 (PAI1), a major inhibitor of fibrinolysis, were involved. Silencing of PAI1 attenuated the anti‐apoptotic effect of human plasma. Furthermore, combined Akt1 and PAI1 silencing attenuated the majority of the anti‐apoptotic effect of human plasma. We conclude that human plasma protects against endothelial cell apoptosis through sustained BAD phosphorylation, which is achieved by, at least in part, a novel interaction between PP1 with PAI1.     

Protein phosphatase PP4 is involved in NHEJ-mediated repair of DNA double-strand breaks

Reversible phosphorylation is an essential posttranslational modification to turn on/off a protein function and to regulate many cellular activities, including DNA repair. A DNA double-strand break (DSB) is the most lethal form of DNA damage and is mainly fixed by the error-prone nonhomologous end joining (NHEJ)-mediated repair and by the high-fidelity homology recombination (HR)-mediated repair. We found previously that protein phosphatase PP4 is required for HR-mediated DSB repair. In this report, we showed that depletion of PP4C by siRNA compromised NHEJ-mediated repair of DSBs induced by the nuclease I-SceI. Both PP4C and its regulatory subunit PP4R2 physically interacted with the chromatin condensation factor KAP1 (KRAB-associated protein 1). Depletion of PP4C led to sustained phosphorylation of KAP1 at Ser824. Conversely, overexpression of PP4C resulted in a decrease of KAP1 phosphorylation. PP4 dephosphorylated pKAP1 in vitro. Inhibition of KAP1 expression resulted in a defect on NHEJ-mediated DSB repair, and co-depletion of PP4c and KAP1 did not have significant synergistic effect on NHEJ-mediated DSB repair. Taken together, our results suggest that PP4C and KAP1 are in the same epistasis group, and PP4 is involved in NHEJ-mediated DSB repair, possibly through regulating the phosphorylation status of KAP1.     

KNL1 Binding to PP1 and Microtubules Is Mutually Exclusive

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Serine/threonine protein phosphatase 5 (PP5) interacts with substrate under heat stress conditions and forms protein complex in Arabidopsis

Protein phosphatase 5 plays a pivotal role in signal transduction in animal and plant cells, and it was previously shown that Arabidopsis protein phosphatase 5 (AtPP5) performs multiple enzymatic activities that are mediated by conformational changes induced by heat shock stress. In addition, transgenic overexpression of AtPP5 gene conferred enhanced heat shock resistance compared with wild-type plant. However, the molecular mechanism underlying this enhanced heat shock tolerance through functional and conformational changes upon heat stress is not clear. In this report, AtPP5 was shown to preferentially interact with its substrate, MDH, under heat stress conditions. In addition, in co-IP analysis, AtPP5 was observed to form a complex with AtHsp90 in Arabidopsis. These results suggest that AtPP5 may enhance thermotolerance via forming multi-chaperone complexes under heat shock conditions in Arabidopsis. Finally, we show that AtPP5 is primarily localized in the cytoplasm of Arabidopsis.     

Antagonistic functions of Par-1 kinase and protein phosphatase 2A are required for localization of Bazooka and photoreceptor morphogenesis in Drosophila

Establishment and maintenance of apical basal cell polarity are essential for epithelial morphogenesis and have been studied extensively using the Drosophila eye as a model system. Bazooka (Baz), a component of the Par-6 complex, plays important roles in cell polarity in diverse cell types including the photoreceptor cells. In ovarian follicle cells, localization of Baz at the apical region is regulated by Par-1 protein kinase. In contrast, Baz in photoreceptor cells is targeted to adherens junctions (AJs). To examine the regulatory pathways responsible for Baz localization in photoreceptor cells, we studied the effects of Par-1 on Baz localization in the pupal retina. Loss of Par-1 impairs the maintenance of AJ markers including Baz and apical polarity proteins of photoreceptor cells but not the establishment of cell polarity. In contrast, overexpression of Par-1 or Baz causes severe mislocalization of junctional and apical markers, resulting in abnormal cell polarity. However, flies with similar overexpression of kinase-inactive mutant Par-1 or unphosphorylatable mutant Baz protein show relatively normal photoreceptor development. These results suggest that dephosphorylation of Baz at the Par-1 phosphorylation sites is essential for proper Baz localization. We also show that the inhibition of protein phosphatase 2A (PP2A) mimics the polarity defects caused by Par-1 overexpression. Furthermore, Par-1 gain-of-function phenotypes are strongly enhanced by reduced PP2A function. Thus, we propose that antagonism between PP2A and Par-1 plays a key role in Baz localization at AJ in photoreceptor morphogenesis.     

Dephosphorylation of Orc2 by protein phosphatase 1 promotes the binding of the origin recognition complex to chromatin

Phosphorylation of Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2–5) from human chromatin and replication origins. Dephosphorylation of the phosphorylated Orc2 by protein phosphatase 1 (PP1) is accompanied by the binding of the dissociated subunits to chromatin. Here we show that PP1 physically interacts with Orc2. The binding of PP1 to Orc2 and the dephosphorylation of Orc2 by PP1 occurred in a cell cycle-dependent manner through an interaction with 119-KSVSF-123, which is the consensus motif for the binding of PP1, of Orc2. The dephosphorylation of Orc2 by PP1 is required for the binding of Orc2 to chromatin. These results support that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin and replication origins for the subsequent round of the cell cycle.     

A novel monoclonal antibody DC63 reveals that inhibitor 1 of protein phosphatase 2A is preferentially nuclearly localised in human brain

Abnormal phosphorylation of tau protein represents one of the major candidate pathological mechanisms leading to Alzheimer's disease (AD) and related tauopathies. Altered phosphorylation status of neuronal tau protein may result from upregulation of tau-specific kinases or from inhibition of tau-specific phosphatases. Increased expression of the protein inhibitor 1 of protein phosphatase 2A (I1PP2A) could therefore indirectly regulate the phosphorylation status of tau. As an important step towards elucidation of the role of I1PP2A in the physiology and pathology of tau phosphorylation, we developed a novel monoclonal antibody, DC63, which recognizes I1PP2A. Specificity of the antibody was examined by mass spectrometry and Western blot. This analysis supports the conclusion that the antibody does not recognize any of the other proteins of the 9-member leucine-rich acidic nuclear phosphoprotein family to which I1PP2A belongs. Immunoblot detection revealed that the inhibitor I1PP2A is expressed throughout the brain, including the hippocampus, temporal cortex, parietal cortex, subcortical nuclei and brain stem. The cerebellum displayed significantly higher levels of expression of I1PP2A than was seen elsewhere in the brain. Imunohistochemical analysis of normal human brain showed that I1PP2A is expressed in both neurons and glial cells and that the protein is preferentially localized to the nucleus. We conclude that the novel monoclonal antibody DC63 could be successfully employed as a mass spectrometry-validated molecular probe that may be used for in vitro and in vivo qualitative and quantitative studies of physiological and pathological pathways involving I1PP2A.     

Clathrin light chain b is capable of affecting potently a major protein phosphatase from microtubules (MT-PP1)

Clathrin light chain (CL) b purified from bovine brain postmicrotubule supernatant and identified by mass spectrometry potently inhibited a catalytic activity of a major protein phosphatase (PP) that was copurified with microtubules and recognized by antiPP1 antibodies. CLb similarly affected the catalytic subunit and holoenzyme of the PP, little inhibiting the activity of PP2A. Although the CLb from clathrin-coated vesicles was several hundredfold weaker than our purified CLb, the CLb in the postmicrotubule supernatant, independent of whether it was sedimentable or soluble, was as active as the purified CLb. Thus CLb may be a potent regulator of the PP.     

A hypermorphic mutation in the protein phosphatase 2C HAB1 strongly affects ABA signaling in Arabidopsis

Protein phosphatases of the 2C family (PP2C) function in the regulation of several signaling pathways from prokaryotes to eukaryotes. In Arabidopsis thaliana, the HAB1 PP2C is a negative regulator of the stress hormone abscisic acid (ABA) signaling. Here, we show that plants expressing a mutant form of HAB1 in which Gly246 was mutated to Asp (G246D) display strong ABA insensitive phenotypes. Our results indicate that the G246D mutation has a hypermorphic rather than a dominant negative effect. The data suggest that this mutation localized in a conserved motif in the PP2C catalytic domain could be used in other PP2Cs to reveal their biological functions.