scholarly journals Phosphorylation-site mutagenesis of the growth-associated protein GAP-43 modulates its effects on cell spreading and morphology.

1993 ◽  
Vol 120 (2) ◽  
pp. 503-512 ◽  
Author(s):  
F Widmer ◽  
P Caroni
Keyword(s):  
Phosphorylation Site ◽  
Growth Cones ◽  
Cell Cortex ◽  
Membrane Association ◽  
Major Protein ◽  
Cell Periphery ◽  
Cortical Actin ◽  
Cortical Cytoskeleton

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane-association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP-43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin-containing cortical cytoskeleton in a regulatable manner.

scholarly journals Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Masayasu Okada ◽  
Yosuke Kawagoe ◽  
Yuta Sato ◽  
Motohiro Nozumi ◽  
Yuya Ishikawa ◽  
...  
Keyword(s):  
Molecular Marker ◽  
Phosphorylation Site ◽  
Axon Growth ◽  
Growth Cones ◽  
Specific Protein ◽  
Phosphorylation Sites ◽  
Quarter Century ◽  
Wide Range

AbstractGAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.

scholarly journals BPGAP1 Interacts with Cortactin and Facilitates Its Translocation to Cell Periphery for Enhanced Cell Migration

2004 ◽  
Vol 15 (6) ◽  
pp. 2873-2883 ◽  
Author(s):  
Bee Leng Lua ◽  
Boon Chuan Low
Keyword(s):  
Cell Migration ◽  
Control Cell ◽  
Actin Binding ◽  
Ionization Mass ◽  
Cell Periphery ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Cell Dynamics

Rho GTPases control cell dynamics during growth and development. They are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). Many GAPs exist with various protein modules, the functions of which largely remain unknown. We recently cloned and identified BPGAP1 as a novel RhoGAP that coordinately regulates pseudopodia and cell migration via the interplay of its BNIP-2 and Cdc42GAP homology, RhoGAP, and the proline-rich domains. To further elucidate the molecular mechanism underlying cell dynamics control by BPGAP1, we used protein precipitations and matrix-assisted laser desorption/ionization mass spectrometry and identified cortactin, a cortical actin binding protein as a novel partner of BPGAP1 both in vitro and in vivo. Progressive deletion studies confirmed that cortactin interacted directly and constitutively with the proline-rich motif 182-PPPRPPLP-189 of BPGAP1 via its Src homology 3 domain. Together, they colocalized to periphery and enhanced cell migration. Furthermore, substitution of prolines at 184 and 186 with alanines abolished their interaction. Consequently, this BPGAP1 mutant failed to facilitate translocation of cortactin to the periphery, and no enhanced cell migration was observed. These results provide the first evidence that a RhoGAP functionally interacts with cortactin and represents a novel determinant in the regulation of cell dynamics.

scholarly journals Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

1996 ◽  
Vol 313 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Françoise LEVAVASSEUR ◽  
Jocelyne LIÉTARD ◽  
Kohei OGAWA ◽  
Nathalie THÉRET ◽  
Peter D. BURBELO ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Basement Membranes ◽  
Major Protein ◽  
Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

scholarly journals Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

2003 ◽  
Vol 23 (8) ◽  
pp. 2953-2968 ◽  
Author(s):  
Ville Hietakangas ◽  
Johanna K. Ahlskog ◽  
Annika M. Jakobsson ◽  
Maria Hellesuo ◽  
Niko M. Sahlberg ◽  
...  
Keyword(s):  
Heat Shock ◽  
Phosphorylation Site ◽  
Stress Granules ◽  
Heat Shock Factor ◽  
Transcriptional Level ◽  
Heat Shock Factor 1 ◽  
Protection Mechanism ◽  
Sumo Modification

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals Regulation of GABAergic synapse formation and plasticity by GSK3β-dependent phosphorylation of gephyrin

2010 ◽  
Vol 108 (1) ◽  
pp. 379-384 ◽  
Author(s):  
Shiva K. Tyagarajan ◽  
Himanish Ghosh ◽  
Gonzalo E. Yévenes ◽  
Irina Nikonenko ◽  
Claire Ebeling ◽  
...  
Keyword(s):  
Synaptic Plasticity ◽  
Hippocampal Neurons ◽  
Glycogen Synthase ◽  
Phosphorylation Site ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Gabaergic Synapse ◽  
Gabaergic Synapses

Postsynaptic scaffolding proteins ensure efficient neurotransmission by anchoring receptors and signaling molecules in synapse-specific subcellular domains. In turn, posttranslational modifications of scaffolding proteins contribute to synaptic plasticity by remodeling the postsynaptic apparatus. Though these mechanisms are operant in glutamatergic synapses, little is known about regulation of GABAergic synapses, which mediate inhibitory transmission in the CNS. Here, we focused on gephyrin, the main scaffolding protein of GABAergic synapses. We identify a unique phosphorylation site in gephyrin, Ser270, targeted by glycogen synthase kinase 3β (GSK3β) to modulate GABAergic transmission. Abolishing Ser270 phosphorylation increased the density of gephyrin clusters and the frequency of miniature GABAergic postsynaptic currents in cultured hippocampal neurons. Enhanced, phosphorylation-dependent gephyrin clustering was also induced in vitro and in vivo with lithium chloride. Lithium is a GSK3β inhibitor used therapeutically as mood-stabilizing drug, which underscores the relevance of this posttranslational modification for synaptic plasticity. Conversely, we show that gephyrin availability for postsynaptic clustering is limited by Ca2+-dependent gephyrin cleavage by the cysteine protease calpain-1. Together, these findings identify gephyrin as synaptogenic molecule regulating GABAergic synaptic plasticity, likely contributing to the therapeutic action of lithium.

Phosphoenolpyruvate carboxylase during grape berry development: protein level, enzyme activity and regulation

2000 ◽  
Vol 27 (3) ◽  
pp. 221 ◽  
Author(s):  
Paraskevi Diakou ◽  
Laurence Svanella ◽  
Philippe Raymond ◽  
Jean-Pierre Gaudillère ◽  
Annick Moing
Keyword(s):  
Malic Acid ◽  
Protein Level ◽  
Phosphoenolpyruvate Carboxylase ◽  
Phosphorylation Site ◽  
Acid Synthesis ◽  
Grape Berry ◽  
Vitis Vinifera L ◽  
Normal Acid

The protein level and regulation of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31, involved in malic acid synthesis) was studied during the fruit development of two grape (Vitis vinifera L.) varieties, ‘Cabernet Sauvignon’ and ‘Gora Chirine’, with berries of normal and low organic acid content, respectively. The protein level and in vitro activity were higher in the low-acid variety than in the normal-acid variety for most stages. In vivo PEPC activity, measured using 14 CO2 labelling, was significantly higher in the low-acid variety than in the normal-acid variety about 1 week before and 1 week after veraison (the day which corresponds to the onset of ripening). However, partitioning into malate was the same for both varieties. Antibodies raised against the N-terminal part of SorghumPEPC recognised the grape berry PEPC, indicating the presence of the consensus phosphorylation site involved in PEPC regulation. PEPC phosphorylation status was estimated by studying sensitivity to pH and malate. Grape berry PEPC appeared more sensitive to low pH and malate during ripening (IC50 malate, 0.2–0.7 mM) compared to during the earlier stages of development (IC50 malate, 1.2–2 mM) for both varieties. Therefore, in the normal-acid variety, PEPC seems to participate in controlling malic acid accumulation but does not seem to control the differences in malic acid concentration observed between the two varieties.

scholarly journals Vimentin provides the mechanical resilience required for amoeboid migration and protection of the nucleus

2019 ◽  
Author(s):  
Luiza Da Cunha Stankevicins ◽  
Marta Urbanska ◽  
Daniel AD. Flormann ◽  
Emmanuel Terriac ◽  
Zahra Mostajeran ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dendritic Cells ◽  
Cell Migration ◽  
Molecular Mechanisms ◽  
Cell Cortex ◽  
Dna Double Strand Breaks ◽  
Nuclear Positioning ◽  
Mechanical Stiffness

AbstractDendritic cells use amoeboid migration through constricted passages to reach the lymph nodes, and this homing function is crucial for immune responses. Amoeboid migration requires mechanical resilience, however, the underlying molecular mechanisms for this type of migration remain unknown. Because vimentin intermediate filaments (IFs) and microfilaments regulate adhesion-dependent migration in a bidirectional manner, we analyzed if they exert a similar control on amoeboid migration. Vimentin was required for cellular resilience, via a joint interaction between vimentin IFs and F-actin. Reduced actin mobility in the cell cortex of vimentin-reduced cells indicated that vimentin promotes Factin subunit exchange and dynamics. These mechano-dynamic alterations in vimentin-deficient dendritic cells impaired amoeboid migration in confined environments in vitro and blocked lymph node homing in mouse experiments in vivo. Correct nuclear positioning is important in confined amoeboid migration both to minimize resistance and to avoid DNA damage. Vimentin-deficiency also led to DNA double strand breaks in the compressed dendritic cells, pointing to a role of vimentin in nuclear positioning. Together, these observations show that vimentin IF-microfilament interactions provide both the specific mechano-dynamics required for dendritic cell migration and the protection the genome needs in compressed spaces.Summary statementVimentin — in joint action with actin — mediates the mechanical stiffness of cells required for amoeboid cell migration through confined spaces and protects the nucleus from DNA damage.

scholarly journals Preclinical Immuno-recognition and Neutralization of Lethality Assessment of a New Polyvalent Antivenom, VINS Snake Venom Antiserum – African IHS®, against Envenomation of Ten African Viperid and Elapid Snakes

2021 ◽  
pp. 25-43
Author(s):  
Djameh, Georgina I. ◽  
Nyarko, Samuel ◽  
Tetteh-Tsifoanya, Mark ◽  
Marfo, Frances M. ◽  
Adjei, Samuel ◽  
...  
Keyword(s):  
Snake Venom ◽  
Sub Saharan Africa ◽  
In Vivo Studies ◽  
Health Concern ◽  
Major Protein ◽  
Snake Species ◽  
Neutralizing Capacity ◽  
Preclinical Efficacy

Snakebite envenomation is a major health concern in developing countries causing significant mortality and morbidity. With over 1.2 million cases annually caused by medically important snake species belonging to the two families Viperidae (Echis spp. and Bitis spp.) and Elapidae (Naja spp. and Dendroaspis spp.). Several antivenoms are being produced and distributed to western sub-Saharan Africa for treatment of envenomation with the absence of preclinical efficacy studies. The present study evaluated the preclinical efficacy of venoms from Echis leucogaster, Echis ocellatus, Bitis arietans, Bitis gabonica, Naja haje, Naja melanoleuca, Naja nigricollis, Dendroaspis jamesoni, Dendroaspis polylepis and Dendroaspis viridis against a polyvalent Snake Venom Antiserum - African IHS (lyophilised), manufactured by VINS Bioproducts Limited (Telangana, India). Our in vitro results showed that, the SVA- AIHS contains antibodies that are capable of recognizing and binding majority of protein components representative of all eight major protein families of venoms of the snake species tested by double immunodiffusion assay and confirmed by western blot. The venom antiserum exhibited high neutralization efficacy against all the viperid and elapid snake species venoms in in vivo studies and confirmed the manufacturer’s recommended neutralization capacity. This is clear evidence that the VINS polyvalent SVA-AIHS batch tested has strong neutralizing capacity and will be useful in treating envenoming by most African viperid and some elapid snake species.

scholarly journals Identification of a Novel Cortactin SH3 Domain-Binding Protein and Its Localization to Growth Cones of Cultured Neurons

1998 ◽  
Vol 18 (10) ◽  
pp. 5838-5851 ◽  
Author(s):  
Yunrui Du ◽  
Scott A. Weed ◽  
Wen-Cheng Xiong ◽  
Trudy D. Marshall ◽  
J. Thomas Parsons
Keyword(s):  
Hippocampal Neurons ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Growth Cones ◽  
Sh3 Domain ◽  
Actin Binding ◽  
Sequence Motifs ◽  
Cortical Actin ◽  
Cytoskeleton Reorganization

ABSTRACT Cortactin is an actin-binding protein that contains several potential signaling motifs including a Src homology 3 (SH3) domain at the distal C terminus. Translocation of cortactin to specific cortical actin structures and hyperphosphorylation of cortactin on tyrosine have been associated with the cortical cytoskeleton reorganization induced by a variety of cellular stimuli. The function of cortactin in these processes is largely unknown in part due to the lack of information about cellular binding partners for cortactin. Here we report the identification of a novel cortactin-binding protein of approximately 180 kDa by yeast two-hybrid interaction screening. The interaction of cortactin with this 180-kDa protein was confirmed by both in vitro and in vivo methods, and the SH3 domain of cortactin was found to direct this interaction. Since this protein represents the first reported natural ligand for the cortactin SH3 domain, we designated it CortBP1 for cortactin-binding protein 1. CortBP1 contains two recognizable sequence motifs within its C-terminal region, including a consensus sequence for cortactin SH3 domain-binding peptides and a sterile alpha motif. Northern and Western blot analysis indicated that CortBP1 is expressed predominately in brain tissue. Immunofluorescence studies revealed colocalization of CortBP1 with cortactin and cortical actin filaments in lamellipodia and membrane ruffles in fibroblasts expressing CortBP1. Colocalization of endogenous CortBP1 and cortactin was also observed in growth cones of developing hippocampal neurons, implicating CortBP1 and cortactin in cytoskeleton reorganization during neurite outgrowth.

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