Phosphorylation-dependent Change Research Articles

Modulation of Structure and Dynamics of Cardiac Troponin by Phosphorylation and Mutations Revealed by Molecular Dynamics Simulations.

Adrenaline acts on β1 receptors in the heart muscle to enhance contractility, increase the heart rate, and increase the rate of relaxation (lusitropy) via activation of the cyclic AMP-dependent protein kinase, PKA. Phosphorylation of serines 22 and 23 in the N-terminal peptide of cardiac troponin I is responsible for lusitropy. Mutations associated with cardiomyopathy suppress the phosphorylation-dependent change. Key parts of troponin responsible for this modulatory system are disordered and cannot be resolved by conventional structural approaches. We performed all-atom molecular dynamics simulations (5 × 1.5 μs runs) of the troponin core (419 amino acids) in the presence of Ca2+ in the bisphosphorylated and unphosphorylated states for both wild-type troponin and the troponin C (cTnC) G159D mutant. PKA phosphorylation affects troponin dynamics. There is significant rigidification of the structure involving rearrangement of the cTnI(1-33)-cTnC interaction and changes in the distribution of the cTnC helix A/B angle, troponin I (cTnI) switch peptide (149-164) docking, and the angle between the regulatory head and ITC arm domains. The familial dilated cardiomyopathy cTnC G159D mutation whose Ca2+ sensitivity is not modulated by cTnI phosphorylation exhibits a structure inherently more rigid than the wild type, with phosphorylation reversing the direction of all metrics relative to the wild type.

Ultrasensitive Change in Nucleosome Binding by Multiple Phosphorylations to the Intrinsically Disordered Region of the Histone Chaperone FACT

Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. We reported previously that the DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites, and the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation (“ultrasensitive response”). In this report, we explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434–624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. We propose that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.

Multiple-Phosphorylation to IDR in the Chromatin Remodeler Fact Shows an ‘Ultrasensitive’ Response in Its Nucleosome Binding

The DNA binding domain of the chromatin remodeler FACT (FAcilitate Chromatin Transcription) comprises acidic and basic intrinsically disordered regions (IDRs). Acidic IDR has ten phosphorylation sites, and they are fully phosphorylated when FACT expressed in the cell. The hyperphosphorylated FACT does not bind to the nucleosome, while the non-phosphorylated form shows the binding. FACT binding ability recovers in a sigmoidal manner according to the number of phosphates in the acidic IDR, which resembles the ‘ultrasensitive’ response previously found for the phosphorylation-dependent change in the interaction between Cdc4 and Sic1 (Mittag et al. PNAS (2008)). This work demonstrates the ultrasensitive response of FACT occurs through the intramolecular interaction between the acidic and basic IDRs in the DNA binding domain of FACT. The ultrasensitive response of FACT, however, does not rely on a large ensemble of the IDR-containing complex as in the Cdc4-Sic1 interaction, but it requires rather restricted conformational dynamics in the interaction between acidic and basic IDRs. With the finding, we propose the other mechanism for the IDR-mediating ultrasensitivity, as an additional function associated explicitly with the IDR.

Inhibition of Calsequestrin Phosphorylation Leads to its Trafficking from Rough Endoplasmic Reticulum

Calsequestrin 2 (CSQ) is a protein in heart cells that concentrates within the sarcoplasmic reticulum (SR), where it incorporates into Ca2+-release complexes. CSQ has a single N-linked glycan that can be used to determine the extent of its trafficking through the secretory system, and multiple C-terminal protein kinase CK2 sensitive phosphorylation sites. Recent data suggests that polymerization of CSQ defines its localization, and leads to its retention within discrete secretory compartments. In these studies, we show that phosphorylation may be responsible for localization of cardiac CSQ within the rough ER of nonmuscle cells and cardiomyocytes. Triple point mutations were made to wild-type canine CSQ (CSQ-WT) phosphorylation sites, either mimicking (CSQ-aPP, S378,382,386E) or inhibiting phosphorylation (CSQ-nPP, S378,382,386A). Overexpression into COS cells showed that, compared to CSQ-WT, mannose trimming of the CSQ glycan increased for CSQ-nPP, reflecting movement out of rough ER; whereas no change was seen for CSQ-aPP. This apparent phosphorylation-dependent change in subcellular localization in COS cells could be observed by confocal immunofluorescence microscopy. The CK2 specific inhibitor tetrabromocinnamic acid (TBCA) blocks phosphorylation of CSQ in vitro by CK2 or by endogenous CSQ kinases with an identical dose response, supporting the identity of CK2 as the CSQ kinase. TBCA (100μM) also inhibited phosphorylation in cultured cells by roughly 80%. Analysis of CSQ overexpressed in these cells showed an effect on CSQ trafficking that was highly similar to the effects of the phosphorylation-site mutant CSQ-nPP. These data support a hypothesis that CSQ phosphorylation by protein kinase CK2 acts to retain cardiac CSQ in rough ER. In cardiomyocytes, growth-dependent phosphorylation of CSQ produces rough ER retention resulting in redistribution of CSQ from junctional SR to the nuclear envelope, the site of IP3-dependent ER Ca2+ storage.

Site of Action of β-Ligands at the Human β1-Adrenoceptor

Antagonist affinity measurements have traditionally been considered important in defining the receptor or receptor subtypes present within cells or tissues. Any change in this value has normally been taken as evidence for the presence of a second receptor. However, highly efficacious ligands induce a time and phosphorylation-dependent change in the beta2-adrenoceptor resulting in 10-fold lower affinity for antagonists. Also the beta1-adrenoceptor is now considered to exist in two different active conformations which are distinguished by their pharmacological properties. In this study, the site of action of a range of beta-agonists and beta-antagonists was determined using the human beta1-adrenoceptor stably expressed in Chinese hamster ovary cells with cyclic AMP response element reporter genes. Adrenaline and noradrenaline were confirmed as having agonist actions via the catecholamine site, whereas all antagonists had higher affinity for the catecholamine rather than secondary site. However, the rank order of affinity for the two sites was different suggesting that they are indeed separate entities. The measurements of antagonist affinity at the catecholamine site, however, were found to depend upon the agonist present. For example, xamoterol, cimaterol, terbutaline, and formoterol agonist responses were more readily antagonized by CGP 20712A[2-hydroxy-5-(2-[{hydroxy-3-(4-[1-methyl-4-trifluoromethyl-2-imidazolyl]phenoxy)propyl}amino]ethoxy)benzamide] than the catecholamine responses themselves. This, however, was not related to agonist efficacy as has previously been reported for the human beta2-adrenoceptor. Therefore, it may be that some agonists (e.g., cimaterol) purely activate the catecholamine site and others purely activate the secondary site (e.g., CGP 12177 [(-)-4-(3-tert-butylamino-2-hydroxypropoxy)-benzimidazol-2-one]), whereas the others (e.g., catecholamines) activate both sites to differing degrees.

Regulation of Nuclear Import by Phosphorylation Adjacent to Nuclear Localization Signals

Many important regulatory proteins, including cell cycle regulators and transcription factors, contain a phosphorylation site within or adjacent to a classic nuclear localization signal (NLS) sequence. Previous studies show that the nuclear localization of these cargoes can be regulated by phosphorylation at these sites. It was hypothesized that this phosphorylation regulates the nuclear import of NLS cargo proteins by modulating the interaction of the cargo with the classic nuclear transport receptor, importin alpha. In this study, we utilize in vitro solution binding assays and in vivo analyses to directly test this model. We demonstrate that mimicking phosphorylation at a site adjacent to an NLS decreases the binding affinity of the NLS for importin alpha. This decrease in cargo affinity for importin alpha correlates with a decrease in nuclear accumulation in vivo. Through these analyses, we show that the cell cycle-dependent nuclear import of the Saccharomyces cerevisiae transcription factor Swi6p correlates with a phosphorylation-dependent change in affinity for importin alpha. Furthermore, we present data using the SV40 NLS to suggest that this form of regulation can be utilized to artificially modulate the nuclear import of a cargo, which is usually constitutively targeted to the nucleus. This work defines one molecular mechanism for regulating nuclear import by the classic NLS-mediated transport pathway.

Activation of the Leukocyte NADPH Oxidase Subunit p47phox by Protein Kinase C. A Phosphorylation-Dependent Change in the Conformation of the C-Terminal End of p47phox

Activation of the Leukocyte NADPH Oxidase Subunit p47<i>^p^h^o^x</i> by Protein Kinase C. A Phosphorylation-Dependent Change in the Conformation of the C-Terminal End of p47<i>^p^h^o^x</i>

Control of the mutagenicity of aromatic amines by protein kinases and phosphatases. I. The protein phosphatase inhibitors okadaic acid and ortho-vanadate drastically reduce the mutagenicity of aromatic amines.

The role of protein kinase C and protein phosphatases was examined in the control of mutagenic metabolites of aromatic amines. Various metabolic activating systems derived from rat liver were treated with: 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C modulator; okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatases (PP1 and PP2A); and ortho-vanadate (OV), an inhibitor of tyrosine phosphatases. TPA used over a wide concentration range (10(-9)-10(-6) M) did not affect the bacterial mutagenicity of the aromatic amines and of the aromatic amide investigated, 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene (2AAF). At the molecular level, TPA did not affect the function of cytochrome P450s 1A1 or 1A2, which are known key factors for the activation and inactivation of aromatic amines/amides. By contrast the OA and OV treatment of rat hepatocytes, rat liver homogenate, fraction S9 and the nuclear fraction drastically reduced (by > 80%) the mutagenicity of the aromatic amines/amide investigated. This is by far the most pronounced change in genotoxicity observed to date via modulation of phosphorylation. Whilst the mutagenicity of the primary toxication product 2-N-OH-acetylaminofluorene (2-N-OH-AAF) in the presence of exogenous activating systems (hepatocytes, S9-fraction, nuclear fraction) was also reduced by OV, OA had no influence. Thus the tyrosine protein phosphatase inhibitor and the serine/threonine protein phosphatase inhibitor influence the genotoxicity of aromatic amines/amides on different levels. Moreover, this shows that the drastic reduction in mutagenicity by OA was due to its influence on a step prior to the presence of the primary toxication product 2-N-OH-AAF. This reduction could be due to changes in the activity of cytochrome P4501A1 and/or 1A2. However, no incorporation of 32P-labelled phosphate from intracellularly prelabelled [32P]-ATP into cytochromes P450 1A1 or 1A2 nor any change in their catalytic activities was observed in the presence of OA. Furthermore, a phosphorylation dependent change in the function of P-glycoprotein (known for its role in the transport of diverse xenobiotic substances and their metabolites) was shown not to contribute to the observed decrease in mutagenicity. Our results reveal an important role for protein phosphatase 1 and/or 2A and tyrosine phosphatase(s) in the control of the genotoxicity of aromatic amines and amides. However, the present study does not distinguish between effects mediated by individual proteins affected by these protein phosphatases.

BDNF is produced in NGF-responsive sensory neurons of rats

Nerve growth factor (NGF) and related members of the neurotrophin gene family are essential for the selective survival and differentiation of specific neurons within the peripheral and central nervous systems. This chapter summarizes several recent studies that suggest a novel role for these trophic factors in the dynamic modulation of synaptic efficacy, both during development and in maturity. The neurotrophin gene family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Three trk family members, trkA, trkB, and trkC, encode essential components of receptors for these neurotrophins. Neurotrophin receptors exist in several isoforms. The full-length, active forms of the trk family are necessary for the normal development and maintenance of specific neuronal populations. To investigate underlying synaptic mechanisms, the BDNF effect has been characterized in whole-cell voltage-clamp experiments. Recordings of excitatory postsynaptic currents (EPSCs) revealed a marked enhancement within 2-3 min of BDNF exposure. BDNF application has elicited a twofold increase in synaptic charge with a time course similar to the increase in action potential activity. Anoother neurotrophin NT-4, ligands at the trkB receptor, has also rapidly increased both the frequency and amplitude of excitatory synaptic currents in hippocampal neurons. The potentiation of synaptic current amplitude, in particular, results from a phosphorylation-dependent change in postsynaptic responsiveness. Neurotrophins have also been shown to enhance synaptic transmission by increasing presynaptic transmitter release. These results suggest that trophic factors may play a central role in the dynamic modulation of synaptic efficacy. Acute modulation by neurotrophins may constitute an intermediary stage, preceding more stable changes that depend on alterations in gene expression and/or protein synthesis.

1627 Quantification of mRNAs for neurotrophins and their receptors in primate visual cortices

Nerve growth factor (NGF) and related members of the neurotrophin gene family are essential for the selective survival and differentiation of specific neurons within the peripheral and central nervous systems. This chapter summarizes several recent studies that suggest a novel role for these trophic factors in the dynamic modulation of synaptic efficacy, both during development and in maturity. The neurotrophin gene family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Three trk family members, trkA, trkB, and trkC, encode essential components of receptors for these neurotrophins. Neurotrophin receptors exist in several isoforms. The full-length, active forms of the trk family are necessary for the normal development and maintenance of specific neuronal populations. To investigate underlying synaptic mechanisms, the BDNF effect has been characterized in whole-cell voltage-clamp experiments. Recordings of excitatory postsynaptic currents (EPSCs) revealed a marked enhancement within 2-3 min of BDNF exposure. BDNF application has elicited a twofold increase in synaptic charge with a time course similar to the increase in action potential activity. Anoother neurotrophin NT-4, ligands at the trkB receptor, has also rapidly increased both the frequency and amplitude of excitatory synaptic currents in hippocampal neurons. The potentiation of synaptic current amplitude, in particular, results from a phosphorylation-dependent change in postsynaptic responsiveness. Neurotrophins have also been shown to enhance synaptic transmission by increasing presynaptic transmitter release. These results suggest that trophic factors may play a central role in the dynamic modulation of synaptic efficacy. Acute modulation by neurotrophins may constitute an intermediary stage, preceding more stable changes that depend on alterations in gene expression and/or protein synthesis.

1319 BNDF is produced in NGF-responsive sensory neurons of rats

Nerve growth factor (NGF) and related members of the neurotrophin gene family are essential for the selective survival and differentiation of specific neurons within the peripheral and central nervous systems. This chapter summarizes several recent studies that suggest a novel role for these trophic factors in the dynamic modulation of synaptic efficacy, both during development and in maturity. The neurotrophin gene family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Three trk family members, trkA, trkB, and trkC, encode essential components of receptors for these neurotrophins. Neurotrophin receptors exist in several isoforms. The full-length, active forms of the trk family are necessary for the normal development and maintenance of specific neuronal populations. To investigate underlying synaptic mechanisms, the BDNF effect has been characterized in whole-cell voltage-clamp experiments. Recordings of excitatory postsynaptic currents (EPSCs) revealed a marked enhancement within 2-3 min of BDNF exposure. BDNF application has elicited a twofold increase in synaptic charge with a time course similar to the increase in action potential activity. Anoother neurotrophin NT-4, ligands at the trkB receptor, has also rapidly increased both the frequency and amplitude of excitatory synaptic currents in hippocampal neurons. The potentiation of synaptic current amplitude, in particular, results from a phosphorylation-dependent change in postsynaptic responsiveness. Neurotrophins have also been shown to enhance synaptic transmission by increasing presynaptic transmitter release. These results suggest that trophic factors may play a central role in the dynamic modulation of synaptic efficacy. Acute modulation by neurotrophins may constitute an intermediary stage, preceding more stable changes that depend on alterations in gene expression and/or protein synthesis.

Neurotrophin Modulation of Hippocampal Synaptic Transmission

Publisher Summary Nerve growth factor (NGF) and related members of the neurotrophin gene family are essential for the selective survival and differentiation of specific neurons within the peripheral and central nervous systems. This chapter summarizes several recent studies that suggest a novel role for these trophic factors in the dynamic modulation of synaptic efficacy, both during development and in maturity. The neurotrophin gene family includes NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Three trk family members, trkA , trkB , and trkC , encode essential components of receptors for these neurotrophins. Neurotrophin receptors exist in several isoforms. The full-length, active forms of the trk family are necessary for the normal development and maintenance of specific neuronal populations. To investigate underlying synaptic mechanisms, the BDNF effect has been characterized in whole-cell voltage-clamp experiments. Recordings of excitatory postsynaptic currents (EPSCs) revealed a marked enhancement within 2-3 min of BDNF exposure. BDNF application has elicited a twofold increase in synaptic charge with a time course similar to the increase in action potential activity. Anoother neurotrophin NT-4, ligands at the trkB receptor, has also rapidly increased both the frequency and amplitude of excitatory synaptic currents in hippocampal neurons. The potentiation of synaptic current amplitude, in particular, results from a phosphorylation-dependent change in postsynaptic responsiveness. Neurotrophins have also been shown to enhance synaptic transmission by increasing presynaptic transmitter release. These results suggest that trophic factors may play a central role in the dynamic modulation of synaptic efficacy. Acute modulation by neurotrophins may constitute an intermediary stage, preceding more stable changes that depend on alterations in gene expression and/or protein synthesis.

Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation.

Ligand activation causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation.

The effect of ligand binding on platelet-derived growth factor (PDGF) receptor conformation was examined using peptide antibodies directed against specific receptor domains. Antiserum 83, which was directed to the receptor's carboxyl terminus (residues 934-951), preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the antibody to the carboxyl terminus. The activated receptor conformation was induced by three different forms of PDGF (AA and BB homodimers and AB heterodimers) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor, sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by adenyl-5'-yl imidodiphosphate, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in Chinese hamster ovary fibroblasts transfected with wild-type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis even though 125I-PDGF binding was normal. These findings show that ligand binding elicits a phosphorylation-dependent change in PDGF receptor conformation that may be important for receptor function.