scholarly journals Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance

2020 ◽  
Vol 94 (16) ◽  
Author(s):  
Timra Gilson ◽  
Sara Culleton ◽  
Fang Xie ◽  
Marsha DeSmet ◽  
Elliot J. Androphy
Keyword(s):  
Posttranslational Modifications ◽  
Phosphorylation Site ◽  
Binding Partners ◽  
Copy Numbers ◽  
C Terminus ◽  
Epithelial Cell Lines ◽  
Matrix Fraction ◽  
Episomal Maintenance ◽  
Tyrosine Phosphorylation Site

ABSTRACT Several serine and threonine residues of the papillomavirus early E2 protein have been found to be phosphorylated. In contrast, only one E2 tyrosine phosphorylation site in BPV-1 (tyrosine 102) and one in HPV-16/31 (tyrosine 138) site have been characterized. Between BPV-1 and HPV-31 E2, 8 of the 11 tyrosines are conserved in the N-terminal domain, suggesting that phosphorylation of tyrosines has an essential role in E2 biology. In this study, we examine the effect of Y102 phosphorylation on HPV-31 E2 biology. Y102 proteins mutated either to the potential phospho-mimetic glutamic acid (Y102E) or to the nonphosphorylated homologue phenylalanine (Y102F) remain nuclear; however, Y102E is more associated with the nuclear matrix fraction. This is consistent with the inability of Y102E to bind TopBP1. Both BPV-1 and HPV-31 Y102E are similar in that neither binds the C terminus of Brd4, but in all other aspects the mutant behaves differently between the two families of papillomaviruses. BPV-1 Y102E was unable to bind E1 and did not replicate in a transient in vitro assay, while HPV-31 Y102E binds E1 and was able to replicate, albeit at lower levels than wild type. To examine the effect of E2 mutations under more native-like infection conditions, a neomycin-selectable marker was inserted into L1/L2 of the HPV-31 genome, creating HPV-31neo. This genome was maintained in every cell line tested for at least 50 days posttransfection/infection. Y102E in both transfection and infection conditions was unable to maintain high episome copy numbers in epithelial cell lines. IMPORTANCE Posttranslational modifications by phosphorylation can change protein activities, binding partners, or localization. Tyrosine 102 is conserved between delta papillomavirus BPV-1 and alpha papillomavirus HPV-31 E2. We characterized mutations of HPV-31 E2 for interactions with relevant cellular binding partners and replication in the context of the viral genome.

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

2020 ◽  
Vol 19 ◽  
Author(s):  
Sumei Li ◽  
Jifeng Zhang ◽  
Jiaqi Zhang ◽  
Jiong Li ◽  
Longfei Cheng ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Phosphorylation Site ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
C Terminus ◽  
Mediator Protein ◽  
Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

scholarly journals Differential phosphorylation signals control endocytosis of GPR15

2017 ◽  
Vol 28 (17) ◽  
pp. 2267-2281 ◽  
Author(s):  
Yukari Okamoto ◽  
Sojin Shikano
Keyword(s):  
Surface Density ◽  
Functional Role ◽  
Control Cell ◽  
Phosphorylation Site ◽  
Hek293 Cells ◽  
C Terminus ◽  
Differential Phosphorylation ◽  
G Protein Coupled

GPR15 is an orphan G protein–coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the β-arrestin– and GPCR kinase–dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis.

scholarly journals Trypsin digestion of the inositol trisphosphate receptor: implications for the conformation and domain organization of the protein

1995 ◽  
Vol 307 (3) ◽  
pp. 859-865 ◽  
Author(s):  
S K Joseph ◽  
S Pierson ◽  
S Samanta
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Soluble Fraction ◽  
Phosphorylation Site ◽  
C Terminus ◽  
Proteolytic Site ◽  
Dependent Protein ◽  
Limited Digestion ◽  
Trisphosphate Receptor

Limited digestion of rat cerebellum microsomal vesicles with trypsin resulted in the proteolysis of the 240 kDa inositol 1,4,5-trisphosphate receptor (IP3R) and the formation of a 94 kDa species that remained membrane-bound and retained immunoreactivity to an antibody raised against the C-terminal sequence of this protein. The appearance of the 94 kDa species was associated with a loss of [3H]IP3 binding sites in the membrane and the appearance of [3H]IP3 binding sites in the soluble fraction. The 94 kDa fragment retained reactivity to biotinylated concanavalin A. In vitro phosphorylation of the IP3R in membranes with cyclic AMP-dependent protein kinase and [gamma-32P]ATP produced an unlabelled 94 kDa fragment after tryptic digestion. According to current models of the cerebellar IP3R this would place the proteolytic site between the phosphorylation site at serine-1755 and the first transmembrane segment of the IP3R. A second antibody raised to amino acids 401-414 in the N-terminal region of the receptor recognizes a 68 kDa fragment released into the soluble fraction after trypsin treatment. The time course of release of the 68 kDa fragment was correlated with the appearance of soluble binding sites, and the fragment was bound by IP3-Affigel resin. A large proportion of the 68 kDa fragment remained associated with the membrane fraction and could be specifically immunoprecipitated from detergent extracts of digested membranes by anti-C-terminus antibody. Our results provide experimental evidence to further localize the ligand binding domain and suggest that regions of the N-terminus and C-terminus may be non-covalently associated.

scholarly journals Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Stephan Tetenborg ◽  
Helen Y. Wang ◽  
Lena Nemitz ◽  
Anne Depping ◽  
Alexsandra B. Espejo ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Binding Affinity ◽  
Protein Microarray ◽  
Pdz Domain ◽  
Phosphorylation Site ◽  
Binding Domain ◽  
Dependent Manner ◽  
C Terminus ◽  
Activity Dependent

Abstract Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14–3–3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.

scholarly journals Phosphorylation and O-GlcNAcylation of the PHF-1 Epitope of Tau Protein Induce Local Conformational Changes of the C-Terminus and Modulate Tau Self-Assembly Into Fibrillar Aggregates

2021 ◽  
Vol 14 ◽  
Author(s):  
François-Xavier Cantrelle ◽  
Anne Loyens ◽  
Xavier Trivelli ◽  
Oliver Reimann ◽  
Clément Despres ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Tau Protein ◽  
Posttranslational Modifications ◽  
Self Assembly ◽  
Critical Role ◽  
Small Peptides ◽  
C Terminus ◽  
The Impact

Phosphorylation of the neuronal microtubule-associated Tau protein plays a critical role in the aggregation process leading to the formation of insoluble intraneuronal fibrils within Alzheimer’s disease (AD) brains. In recent years, other posttranslational modifications (PTMs) have been highlighted in the regulation of Tau (dys)functions. Among these PTMs, the O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates Tau phosphorylation and aggregation. We here focus on the role of the PHF-1 phospho-epitope of Tau C-terminal domain that is hyperphosphorylated in AD (at pS396/pS404) and encompasses S400 as the major O-GlcNAc site of Tau while two additional O-GlcNAc sites were found in the extreme C-terminus at S412 and S413. Using high resolution NMR spectroscopy, we showed that the O-GlcNAc glycosylation reduces phosphorylation of PHF-1 epitope by GSK3β alone or after priming by CDK2/cyclin A. Furthermore, investigations of the impact of PTMs on local conformation performed in small peptides highlight the role of S404 phosphorylation in inducing helical propensity in the region downstream pS404 that is exacerbated by other phosphorylations of PHF-1 epitope at S396 and S400, or O-GlcNAcylation of S400. Finally, the role of phosphorylation and O-GlcNAcylation of PHF-1 epitope was probed in in-vitro fibrillization assays in which O-GlcNAcylation slows down the rate of fibrillar assembly while GSK3β phosphorylation stimulates aggregation counteracting the effect of glycosylation.

scholarly journals Casein Kinase 2 Binds to the C Terminus of Na+/H+ exchanger 3 (NHE3) and Stimulates NHE3 Basal Activity by Phosphorylating a Separate Site in NHE3

2008 ◽  
Vol 19 (9) ◽  
pp. 3859-3870 ◽  
Author(s):  
Rafiquel Sarker ◽  
Mads Grønborg ◽  
Boyoung Cha ◽  
Sachin Mohan ◽  
Yueping Chen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phosphorylation Site ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Kinase Assay ◽  
Phosphatase Inhibitors ◽  
C Terminus ◽  
Separate Site ◽  
A Site

Na+/H+ exchanger 3 (NHE3) is the epithelial-brush border isoform responsible for most intestinal and renal Na+ absorption. Its activity is both up- and down-regulated under normal physiological conditions, and it is inhibited in most diarrheal diseases. NHE3 is phosphorylated under basal conditions and Ser/Thr phosphatase inhibitors stimulate basal exchange activity; however, the kinases involved are unknown. To identify kinases that regulate NHE3 under basal conditions, NHE3 was immunoprecipitated; LC-MS/MS of trypsinized NHE3 identified a novel phosphorylation site at S719 of the C terminus, which was predicted to be a casein kinase 2 (CK2) phosphorylation site. This was confirmed by an in vitro kinase assay. The NHE3-S719A mutant but not NHE3-S719D had reduced NHE3 activity due to less plasma membrane NHE3. This was due to reduced exocytosis plus decreased plasma membrane delivery of newly synthesized NHE3. Also, NHE3 activity was inhibited by the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole DMAT when wild-type NHE3 was expressed in fibroblasts and Caco-2 cells, but the NHE3-S719 mutant was fully resistant to DMAT. CK2 bound to the NHE3 C-terminal domain, between amino acids 590 and 667, a site different from the site it phosphorylates. CK2 binds to the NHE3 C terminus and stimulates basal NHE3 activity by phosphorylating a separate single site on the NHE3 C terminus (S719), which affects NHE3 trafficking.

scholarly journals A single tyrosine phosphorylation site in cortactin is important for filopodia formation in neuronal growth cones

2019 ◽  
Vol 30 (15) ◽  
pp. 1817-1833 ◽  
Author(s):  
Yuan Ren ◽  
Yingpei He ◽  
Sherlene Brown ◽  
Erica Zbornik ◽  
Michael J. Mlodzianoski ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Leading Edge ◽  
Growth Cones ◽  
Neuronal Growth ◽  
Src Tyrosine Kinase ◽  
Actin Organization ◽  
Neuronal Growth Cones ◽  
Tyrosine Phosphorylation Site

Cortactin is a Src tyrosine phosphorylation substrate that regulates multiple actin-related cellular processes. While frequently studied in nonneuronal cells, the functions of cortactin in neuronal growth cones are not well understood. We recently reported that cortactin mediates the effects of Src tyrosine kinase in regulating actin organization and dynamics in both lamellipodia and filopodia of Aplysia growth cones. Here, we identified a single cortactin tyrosine phosphorylation site (Y499) to be important for the formation of filopodia. Overexpression of a 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of a 499E phospho-mimetic mutant increased filopodia length. Using an antibody against cortactin pY499, we showed that tyrosine-phosphorylated cortactin is enriched along the leading edge. The leading edge localization of phosphorylated cortactin is Src2-dependent, F-actin–independent, and important for filopodia formation. In vitro kinase assays revealed that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro. Finally, we provide evidence that Arp2/3 complex acts downstream of phosphorylated cortactin to regulate density but not length of filopodia. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation.

scholarly journals XIAP controls RIPK2 signaling by preventing its deposition in speck-like structures

2019 ◽  
Author(s):  
Kornelia Ellwanger ◽  
Christine Arnold ◽  
Selina Briese ◽  
Ioannis Kienes ◽  
Jens Pfannstiel ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Complex Formation ◽  
Posttranslational Modifications ◽  
Molecular Structures ◽  
Host Cells ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Enteropathogenic Bacteria ◽  
Binding Partners

ABSTRACTThe receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events.Recently, it was shown that RIPK2 forms higher order molecular structuresin vitro, which were proposed to activate signaling. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depends on the CARD of NOD1 or NOD2. Complex formation upon activation was dependent on RIPK2 autophosphorylation at Y474 and influenced by phosphorylation at S176. Inhibition of activity of the cIAP protein XIAP induced spontaneous complex formation of RIPK2 but blocked NOD1-dependet NF-κB activation. Using immunoprecipitation, we identified 14-3-3 proteins as novel binding partners of non-activated RIPK2, whereas complexed RIPK2 was bound by the prohibitin proteins Erlin-1 and Erlin-2.Taken together, our work reveals novel roles of XIAP, 14-3-3 and Erlin proteins in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling.

Targeting the Insulin-Like Growth Factor-I Receptor (IGF-IR) in Multiple Myeloma Cells Using Selective IGF-IR Tyrosine Kinase Inhibitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 639-639 ◽  
Author(s):  
Thomas Stromberg ◽  
Simon Ekman ◽  
Leonard Girnita ◽  
Johan Lennartsson ◽  
Ulf Hellman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Rpmi 8226 ◽  
Β Chain ◽  
Tyrosine Phosphorylation Site ◽  
Rtk Inhibitors

Abstract In multiple myeloma (MM) emerging evidence suggest the IGF-1R as an important mediator of tumor cell survival and thus resistance to cytotoxic therapy. Since IGF-1R is not an absolute requirement for maintenance of normal cell homeostasis, interfering with IGF-1R signaling at the receptor tyrosine kinase (RTK) level represents an attractive strategy to improve anti-cancer treatment. However, most IGF-1 RTK inhibitors do not fully discriminate between the IGF-1 RTK and the insulin RTK, i.e. are diabetogenic. Recently, members of the cyclolignan family have been shown to selectively inhibit the phosphorylation of the IGF-1R β-chain without down regulating the RTK activity of the insulin R1. The effect of two of these compounds, picropodophyllin (PPP) and deoxypodophyllotoxin (DPPT), was studied in vitro using a panel of nine MM cell lines and primary tumor cells from MM patients, and in vivo using the 5TMM mouse MM model2. Both IGF-1 RTK inhibitors effectively inhibited growth in all MM cell lines providing increased apoptosis and cell cycle arrest in the G2/M-phase. Notably, the two drug-resistant subclones of the MM cell line RPMI 8226/Dox40 (doxorubicin) and RPMI 8226/LR5 (melphalan), were highly sensitive to the IGF-1 RTK inhibitors. In addition, PPP and DPPT showed inhibitory effects on primary MM cells cultured in vitro with bone marrow stromal cells. Inhibition of the IGF-1 RTK with PPP has been shown to be non-competitive with ATP suggesting interference with the IGF-1R at the substrate level1. To identify tyrosine phosphorylation site(s) on the IGF-1R β-chain potentially regulated by the IGF-1 RTK inhibitors, IGF-1R extracted from RPMI 8226 cells was analyzed by tryptic phosphopeptide mapping following 32P-labeling and treatment with PPP or DPPT. Cleaved phosphopeptides were separated by thin layer chromatography and analyzed by a modified radio-Edman degradation. The results show that the IGF-1 RTK inhibitors do not down regulate any specific tyrosine phosphorylation site of the IGF-1R in MM cells. In vitro kinase assay suggests that PPP/DPPT-induced inhibition of the IGF-1R instead is conducted via a general down regulation of the tyrosine kinase activity of the receptor. Extraction of tyrosine phosphorylated proteins followed by electrophoresis and mass spectrometric analysis revealed additional signaling molecules affected by treatment with the IGF-1 RTK inhibitors e.g. impaired tyrosine phosphorylation of CDK1/cdc2, a cell cycle associated protein known as a potent regulator of apoptosis. Taken together, we show that treatment of MM cells with selective IGF-1 RTK inhibitors decreases survival/proliferation and affects the function of crucial intracellular signaling proteins, thus emphasizing the pivotal role for IGF-1R signaling in MM.

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