Phosphorylation-dependent Process Research Articles

PP2A-B55SUR-6 promotes nuclear envelope breakdown in C. elegans embryos

Nuclear envelope (NE) disassembly during mitosis is critical to ensure faithful segregation of the genetic material. NE disassembly is a phosphorylation-dependent process wherein mitotic kinases hyper-phosphorylate lamina and nucleoporins to initiate nuclear envelope breakdown (NEBD). In this study, we uncover an unexpected role of the PP2A phosphatase B55SUR-6 in NEBD during the first embryonic division of Caenorhabditis elegans embryo. B55SUR-6 depletion delays NE permeabilization and stabilizes lamina and nucleoporins. As a result, the merging of parental genomes and chromosome segregation is impaired. NEBD defect upon B55SUR-6 depletion is not due to delayed mitotic onset or mislocalization of mitotic kinases. Importantly, we demonstrate that microtubule-dependent mechanical forces synergize with B55SUR-6 forefficient NEBD. Finally, our data suggest that the lamin LMN-1 is likely a bona fide target of PP2A-B55SUR-6. These findings establish a model highlighting biochemical crosstalk between kinases, PP2A-B55SUR-6 phosphatase, and microtubule-generated mechanical forces in timely NE dissolution.

TrkB-ICD Fragment, Originating From BDNF Receptor Cleavage, Is Translocated to Cell Nucleus and Phosphorylates Nuclear and Axonal Proteins.

The signaling of brain-derived neurotrophic factor (BDNF) has been suggested to be impaired in Alzheimer’s disease (AD), which may compromise the function of BDNF upon neuronal activity and survival. Accordingly, decreased levels of BDNF and its tropomyosin-receptor kinase B-full-length (TrkB-FL) have been detected in human brain samples of AD patients. We have previously found that neuronal exposure to amyloid-β (Aβ) peptide, a hallmark of AD, leads to calpain overactivation and subsequent TrkB-FL cleavage leading to decreased levels of TrkB-FL and the generation of two new fragments: a membrane-bound truncated receptor (TrkB-T′) and an intracellular fragment (TrkB-ICD). Importantly, we identified this TrkB-FL cleavage and TrkB-ICD presence in human brain samples, which indicates that this molecular mechanism contributes to the loss of BDNF signaling in humans. The exact role of this TrkB-ICD fragment is, however, unknown. Here, we used a human neuroglioma cell line and rat cortical primary neuronal cultures to track TrkB-ICD intracellularly. Our data show that TrkB-ICD is a relatively stable fragment that accumulates in the nucleus over time, through a phosphorylation-dependent process. We also found that TrkB-ICD has tyrosine kinase activity, inducing the phosphorylation of nuclear and axonal proteins. These findings suggest that TrkB-ICD may lead to a dysregulation of the activity of several proteins, including proteins in the nucleus, to where TrkB-ICD migrates. Since TrkB-ICD is formed by Aβ peptide-induced cleavage of TrkB-FL, the present data highlights a new mechanism that may have a role in AD pathophysiology.

Divergent Distribution of the Sensor Kinase CosS in Non-Thermotolerant Campylobacter Species and Its Functional Incompatibility with the Response Regulator CosR of Campylobacter jejuni

Two-component signal transduction systems are commonly composed of a sensor histidine kinase and a cognate response regulator, modulating gene expression in response to environmental changes through a phosphorylation-dependent process. CosR is an OmpR-type response regulator essential for the viability of Campylobacter jejuni, a major foodborne pathogenic species causing human gastroenteritis. Although CosR is a response regulator, its cognate sensor kinase has not been identified in C. jejuni. In this study, DNA sequence analysis of the cosR flanking regions revealed that a gene encoding a putative sensor kinase, which we named cosS, is prevalent in non-thermotolerant Campylobacter spp., but not in thermotolerant campylobacters. Phosphorylation assays indicated that C. fetus CosS rapidly autophosphorylates and then phosphorylates C. fetus CosR, suggesting that the CosRS system constitutes a paired two-component signal transduction system in C. fetus. However, C. fetus CosS does not phosphorylate C. jejuni CosR, suggesting that CosR may have different regulatory cascades between thermotolerant and non-thermotolerant Campylobacter species. Comparison of CosR homolog amino acid sequences showed that the conserved phosphorylation residue (D51), which is present in all non-thermotolerant Campylobacter spp., is absent from the CosR homologs of thermotolerant Campylobacter species. However, C. jejuni CosR was not phosphorylated by C. fetus CosS even after site-directed mutagenesis of N51D, implying that C. jejuni CosR may possibly function phosphorylation-independently. In addition, the results of cosS mutational analysis indicated that CosS is not associated with the temperature dependence of the Campylobacter spp. despite its unique divergent distribution only in non-thermotolerant campylobacters. The findings in this study strongly suggest that thermotolerant and non-thermotolerant Campylobacter spp. have different signal sensing mechanisms associated with the CosR regulation.

Structure of an Atypical Orphan Response Regulator Protein Supports a New Phosphorylation-independent Regulatory Mechanism

Two-component signal transduction systems, commonly found in prokaryotes, typically regulate cellular functions in response to environmental conditions through a phosphorylation-dependent process. A new type of response regulator, hp1043 (HP-RR) from Helicobacter pylori, has been recently identified. HP-RR is essential for cell growth and does not require the well known phosphorelay scheme. Unphosphorylated HP-RR binds specifically to its own promoter (P(1043)) and autoregulates the promoter of the tlpB gene (P(tlpB)). We have determined the structure of HP-RR by NMR and x-ray crystallography, revealing a symmetrical dimer with two functional domains. The molecular topology resembles that of the OmpR/PhoB subfamily, however, the symmetrical dimer is stable even in the unphosphorylated state. The dimer interface, formed by three secondary structure elements (alpha4-beta5-alpha5), resembles that of the active, phosphorylated forms of ArcA and PhoB. Several conserved residues of the HP-RR dimeric interface deviate from the OmpR/PhoB subfamily, although there are similar salt bridges and hydrophobic patches within the interface. Our findings reveal how a new type of response regulator protein could function as a cell growth-associated regulator in the absence of post-translational modification.

G Protein-coupled Receptor Kinase and β-Arrestin-mediated Desensitization of the Angiotensin II Type 1A Receptor Elucidated by Diacylglycerol Dynamics

Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

Epstein-Barr Virus Protein Kinase BGLF4 Is a Virion Tegument Protein That Dissociates from Virions in a Phosphorylation-Dependent Process and Phosphorylates the Viral Immediate-Early Protein BZLF1

Epstein-Barr virus (EBV) BGLF4 is a viral protein kinase that is expressed in the lytic phase of infection and is packaged in virions. We report here that BGLF4 is a tegument protein that dissociates from the virion in a phosphorylation-dependent process. We also present evidence that BGLF4 interacts with and phosphorylates BZLF1, a key viral regulator of lytic infection. These conclusions are based on the following observations. (i) In in vitro tegument release assays, a significant fraction of BGLF4 was released from virions in the presence of physiological NaCl concentrations. (ii) Addition of physiological concentrations of ATP and MgCl(2) to virions enhanced BGLF4 release, but phosphatase treatment of virions significantly reduced BGLF4 release. (iii) A recombinant protein containing a domain of BZLF1 was specifically phosphorylated by purified recombinant BGLF4 in vitro, and BGLF4 altered BZLF1 posttranslational modification in vivo. (iv) BZLF1 was specifically coimmunoprecipitated with BGLF4 in 12-O-tetradecanoylphorbol-13-acetate-treated B95-8 cells and in COS-1 cells transiently expressing both of these viral proteins. (v) BGLF4 and BZLF1 were colocalized in intranuclear globular structures, resembling the viral replication compartment, in Akata cells treated with anti-human immunoglobulin G. Our results suggest that BGLF4 functions not only in lytically infected cells by phosphorylating viral and cellular targets but also immediately after viral penetration like other herpesvirus tegument proteins.

Are RB proteins a potential substrate of Pin1 in the regulation of the cell cycle?

RB family members are post-transductionally regulated proteins and phosphorylation at Ser/Thr residues leads to their gradual inactivation. Cyclin/cdk complexes are mainly responsible for the regulation of these pocket proteins, which is crucial for release of E2F factor. Despite the fact that E2F release is a phosphorylation-dependent process, it is still not evident how phosphorylation physically determines the shift from the active to the inactive feature of RB molecules. We would like to put forward the hypothesis that Pin1 is involved in RB proteins phosphorylation and E2F release, suggesting an additional post-translational level of control on this family of molecules.

Calcium-dependent Dephosphorylation Mediates the Hyperosmotic and Lysophosphatidic Acid-dependent Inhibition of Natriuretic Peptide Receptor-B/Guanylyl Cyclase-B

C-type natriuretic peptide binding to natriuretic peptide receptor-B (NPR-B) stimulates cGMP synthesis, which regulates vasorelaxation, cell proliferation, and bone growth. Here, we investigated the mechanistic basis for hyperosmotic and lysophosphatidic acid-dependent inhibition of NPR-B. Whole cell cGMP measurements and guanylyl cyclase assays indicated that acute hyperosmolarity decreased NPR-B activity in a reversible, concentration- and time-dependent manner, whereas chronic exposure had no effect. Acute hyperosmolarity elevated intracellular calcium in a concentration-dependent fashion that paralleled NPR-B desensitization. A calcium chelator, but not a protein kinase C inhibitor, blocked both calcium elevations and desensitization. Hyperosmotic medium stimulated NPR-B dephosphorylation, and the receptor was rapidly rephosphorylated and resensitized when the hypertonic media was removed. Lysophosphatidic acid also inhibited NPR-B in a calcium- and phosphorylation-dependent process, consistent with calcium being a universal regulator of NPR-B. The absolute requirement of dephosphorylation in this process was demonstrated by showing that a receptor with glutamates substituted at all known NPR-B phosphorylation sites is unresponsive to hyperosmotic stimuli. This is the first study to measure the phosphorylation state of an endogenous guanylyl cyclase and to link intracellular calcium elevations with its dephosphorylation.

Immunoreceptor tyrosine-based activation motif phosphorylation during engulfment of Neisseria gonorrhoeae by the neutrophil-restricted CEACAM3 (CD66d) receptor.

Gonorrhea is characterized by a purulent urethral or cervical discharge consisting primarily of neutrophils associated with Neisseria gonorrhoeae. These interactions are facilitated by gonococcal colony opacity-associated (Opa) protein binding to host cellular CEACAM receptors. Of these, CEACAM3 is restricted to neutrophils and contains an immunoreceptor tyrosine-based activation motif (ITAM) reminiscent of that found within certain phagocytic Fc receptors. CEACAM3 was tyrosine phosphorylated by a Src family kinase-dependent process upon infection by gonococci expressing CEACAM-specific Opa proteins. This phosphorylation was necessary for efficient bacterial uptake; however, a less efficient uptake process became evident when kinase inhibitors or mutagenesis of the ITAM were used to prevent phosphorylation. Ligated CEACAM3 was recruited to a cytoskeleton-containing fraction, intense foci of polymerized actin were evident where bacteria attached to HeLa-CEACAM3, and disruption of polymerized actin by cytochalasin D blocked all bacterial uptake by these cells. These data support a model whereby CEACAM3 can mediate the Opa-dependent uptake of N. gonorrhoeae via either an efficient, ITAM phosphorylation-dependent process that resembles phagocytosis or a less efficient, tyrosine phosphorylation-independent mechanism.

C-Mos Proteolysis Is Independent of the Ca2+ Rise Induced by 6-DMAP in Xenopus Oocytes

In Xenopus oocytes, metaphase II arrest is due to a cytostatic factor (CSF) that involves c-Mos, maintaining a high MPF (cdk1/cyclin B) activity in the cell. At fertilization, a rise in intracellular calcium triggers the proteolysis of both cyclin B and c-Mos. The kinase inhibitor 6-dimethylaminopurine (6-DMAP) is also able to release matured Xenopus oocytes from metaphase II block. This is characterized by c-Mos proteolysis without degradation of cyclin B. We hypothesized that 6-DMAP induced an increase in intracellular calcium. Using the calcium-sensitive fluorescent dye Fura-2, we observed a systematic increase in intracellular calcium following 6-DMAP application. In matured oocytes previously microinjected with the calcium chelator BAPTA, no calcium changes occurred after 6-DMAP addition; however, c-Mos was still proteolysed. In oocytes at the GVBD stage, c-Mos proteolysis occurred in response to 6-DMAP but not to calcium ionophore treatment. We suggest that c-Mos proteolysis is rather controlled by a phosphorylation-dependent process.

Relocation of annexin V to platelet membranes is a phosphorylation-dependent process.

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with agents that increase intracellular [Ca2+] induces the relocation of annexin V to membranes, and that this annexin V may be binding to a 50 kDa protein located within platelet membranes. We report here, using an in vitro reconstitution system, that the relocation of annexin V to membranes is enhanced by ATP. We also demonstrate that when adenosine 5'-[gamma-thio]-triphosphate, which can replace ATP in phosphorylation reactions, is substituted for ATP, the amount of annexin V that binds to membranes is further increased. In separate experiments using intact cells, we show that the protein phosphatase inhibitor okadaic acid mimics the action of the physiological agonist thrombin, in that it induces annexin V to bind to membranes and that the addition of the protein kinase inhibitor staurosporine inhibits A23187-induced relocation of annexin V. In addition, alkaline phosphatase, when added to isolated membranes, was found to remove endogenous annexin V from the membranes. Furthermore, immunoprecipitation of 33P-labelled proteins indicated that annexin V may form a multi-protein complex including phosphoproteins of 25, 50 and 83 kDa. Taken together these observations suggest that, following physiological activation, the phosphorylation of one or more proteins is responsible for the tight association of annexin V with platelet membranes and the subsequent regulation of membrane localized processes.

Xnf7 Functions in Dorsal–Ventral Patterning of theXenopusEmbryo

Xenopusnuclear factor 7 (xnf7) is a maternally expressed nuclear protein that is retained in the cytoplasm from oocyte maturation until the midblastula transition (MBT). Mutations of the xnf7 phosphorylation sites to glutamic acids (dnxnf7) resulted in the retention of the endogenous protein in the cytoplasm past the MBT, indicating that cytoplasmic retention is a phosphorylation dependent process. In addition, dnxnf7 acted as a dominant negative mutant by keeping the endogenous xnf7 protein in the cytoplasm past the MBT. Overexpression of dnxnf7 in future dorsal blastomeres resulted in a ventralized or posteriorized phenotype in which the embryos lacked anterior structures, while overexpression in ventral blastomeres resulted in dorsalized embryos. dnxnf7 also affected the expression of both dorsal and ventral mesodermal markers. These data suggest that xnf7 functions in dorsal/ventral patterning and that the movement of the protein from the cytoplasm to the nucleus at the MBT is critical for the execution of a genetic program conferring a dorsal or ventral identity to the mesoderm.

Binding of activated cyclosome to p13(suc1). Use for affinity purification.

Previous studies have indicated that a approximately 1,500-kDa complex, designated the cyclosome or anaphase-promoting complex, has a regulated cyclin-ubiquitin ligase activity that targets cyclin B for degradation at the end of mitosis. The cyclosome is inactive in the interphase of the embryonic cell cycle and is converted to the active form in late mitosis in a phosphorylation-dependent process initiated by protein kinase Cdc2-cyclin B. We show here that the active, phosphorylated form of the cyclosome from clam oocytes binds to p13(suc1), a protein known to associate with Cdc2. The following evidence indicates that the binding of the cyclosome to p13(suc1) is not mediated via the Cdc2-cyclin B complex: (a) activated cyclosome binds to p13(suc1)-Sepharose following its separation from Cdc2-cyclin B by gel filtration chromatography; (b) cyclosome from interphase extracts, activated by a kinase in which cyclin B has been replaced by an N-terminally truncated derivative fused to glutathione S-transferase, binds well to p13(suc1)-Sepharose but not to glutathione-agarose. An alternative possibility, that the phosphorylated cyclosome binds directly to a phosphate-binding site of p13(suc1), is supported by the observation that the cyclosome is efficiently eluted from p13(suc1)-Sepharose by phosphate-containing compounds. This information was utilized to develop a procedure for the affinity purification of the cyclosome. A factor abundant in the fraction not adsorbed to p13(suc1)-Sepharose stimulates the activity of purified cyclosome. It is suggested that binding of Suc1 may have a role in the regulation of cyclosome activity.

Modulation of Ca 2+ channels by α2A-and α2A-adrenoceptors in vascular myocytes: Involvement of different transduction pathways

Using subtype-selective agonists and antagonists, and antibodies directed against phosphatidylinositol and G-proteins, it has been shown in single myocytes of rat portal vein that both α 1A-and α 2A-adrenoreceptors modulate Ca 2+ channels through two distinct transduction pathways. α 1A-adrenoceptors couple with a G q G 11 protein to activate a phospholipase C (PLC) which hydrolyses phosphatidylinositol to generate inositol 1,4,5-trisphosphate (InsP 3) and diacylglycerol (DAG). InsP 3 releases intracellular stored Ca 2+ as evidenced by microspectrofluorimetry with Fura-2. The large and transient increase in [Ca 2+] i activates chloride channels leading to a membrane depolarization that opens voltage-gated Ca 2+ channels. In addition, DAG activates transiently protein kinase C (PKC) which increases the opening probability of Ca 2+ channels through a phosphorylation-dependent process. α- 2A-adrenoceptors do not induce Ca 2+ release from intracellular stores but promote sustained Ca 2+ influx through voltage-gated Ca 2+ channels. The coupling involves a G 1-protein and activation of PKC by DAG. These two transduction pathways may be involved in the physiological action of noradrenaline in vascular smooth muscles.

Nuclear ion channels in cardiac myocytes.

The paradigm that nucleocytoplasmic transport of ions occurs without a diffusional barrier has been challenged by the recent demonstration with patch-clamp techniques of the existence of ion channels in the nuclear envelope of murine zygotes and hepatocytes. This report demonstrates the existence of nuclear ion channels (NIC) in murine ventricular cardiac myocytes. NIC conductance (gamma), calculated from current histogram peaks, was 106-532 pS at 22-36 degrees C. In nucleus-attached patches, replacement of cytoplasmic K+ with Na+ reduced NIC activity within 30 s, suggesting that intranuclear-delimited mechanisms mediate this phenomenon. In excised, inside-out patches K+ was as permeable as Na+ through NIC. NIC activity was observed in 0-4 mM Mg2+ and/or ATP2-, with or without 0-1 mM Ca2+, indicating a minor direct role of these ions. However, in non-responsive excised inside-out patches, NIC activity appeared when the catalytic subunit of the cAMP-dependent protein kinase was applied to the nucleoplasmic side of the patch, in the presence of Mg2+ and ATP2-, indicating an important role for phosphorylation-dependent process(es) in NIC function--an observation supported by the depressing effects of protein kinase inhibitor on responsive NIC. The concept that nucleopore complexes are solely responsible for nucleocytoplamic transport leads to the speculation that these structures are the physical substrate for NIC.

Effects of purine nucleotides on the binding of [3H]cyclopentyladenosine to adenosine A-1 receptors in rat brain membranes.

Adenine nucleotides displace the binding of the selective adenosine A-1 receptor ligand [3H]cyclopentyladenosine (CPA) to rat brain membranes in a concentration-dependent manner, with the rank order of activity being ATP greater than ADP greater than AMP. Binding was also displaced by GTP, ITP, adenylylimidodiphosphate (AppNHp), 2-methylthioATP, and the beta-gamma-methylene isostere of ATP, but was unaffected by the alpha-beta-methylene isosteres of ADP and ATP, and UTP. At ATP concentrations greater than 100 microM, the inhibitory effects on CPA binding were reversed, until at 2 mM ATP, specific binding of CPA was identical to that seen in controls. Concentrations of ATP greater than 10 mM totally inhibited specific binding. Inclusion of the catabolic enzyme adenosine deaminase in the incubation medium abolished the inhibitory effects of ATP, indicating that these were due to adenosine formation, presumably due to ectonucleotidase activity. The inhibitory effects were also attenuated by the alpha-beta-methylene isostere of ATP, an ectonucleotidase inhibitor. Adenosine deaminase, alpha-beta-methylene ATP (100 microM), and beta-gamma-methylene ATP (100 microM) had no effect on the "stimulatory" phase of binding, although GTP (100 microM) slightly attenuated it. Comparison of the binding of [3H]CPA in the absence and presence of 2 mM ATP by saturation analysis showed that the KD and apparent Bmax values were identical. Examination of the pharmacology of the control and "ATP-dependent" CPA binding sites showed slight changes in binding of adenosine agonists and antagonists. The responses observed with high concentrations of ATP were not observed with GTP, AppNHp, the chelating agents EDTA and EGTA, or inorganic phosphate. The divalent cations Mg2+ and Ca2+ at 10 mM attenuated the stimulatory actions of high (2 mM) concentrations of ATP, whereas EGTA and EDTA (10 mM) enhanced the "stimulatory" actions of ATP. EDTA (10 mM) abolished the inhibitory effects of ATP, indicating a specific dependence on Mg2+ for the inhibitory response. The effects of ATP on [3H]CPA binding were reversible for antagonists but not agonists. The mechanism by which ATP reverses its own inhibitory action on adenosine A-1 radioligand binding is unclear, and from the observed actions of the divalent cations and chelating agents probably does not involve a phosphorylation-dependent process.