The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network

1998 ◽  
Vol 111 (22) ◽  
pp. 3333-3346 ◽  
Author(s):  
O. Gavet ◽  
S. Ozon ◽  
V. Manceau ◽  
S. Lawler ◽  
P. Curmi ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Intracellular Signaling ◽  
Splice Variants ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Dependent Manner ◽  
Microtubule Depolymerization ◽  
Microtubule Network

Stathmin is a small regulatory phosphoprotein integrating diverse intracellular signaling pathways. It is also the generic element of a protein family including the neural proteins SCG10, SCLIP, RB3 and its two splice variants RB3′ and RB3″. Stathmin itself was shown to interact in vitro with tubulin in a phosphorylation-dependent manner, sequestering free tubulin and hence promoting microtubule depolymerization. We investigated the intracellular distribution and tubulin depolymerizing activity in vivo of all known members of the stathmin family. Whereas stathmin is not associated with interphase microtubules in HeLa cells, a fraction of it is concentrated at the mitotic spindle. We generated antisera specific for stathmin phosphoforms, which allowed us to visualize the regulation of phosphorylation-dephosphorylation during the successive stages of mitosis, and the partial localization of stathmin phosphorylated on serine 16 at the mitotic spindle. Results from overexpression experiments of wild-type and novel phosphorylation site mutants of stathmin further suggest that it induces depolymerization of interphase and mitotic microtubules in its unphosphorylated state but is inactivated by phosphorylation in mitosis. Phosphorylation of mutants 16A25A and 38A63A on sites 38 and 63 or 16 and 25, respectively, was sufficient for the formation of a functional spindle, whereas mutant 16A25A38A63E retained a microtubule depolymerizing activity. Transient expression of each of the neural phosphoproteins of the stathmin family showed that they are at least partially associated to the Golgi apparatus and not to other major membrane compartments, probably through their different NH2-terminal domains, as described for SCG10. Most importantly, like stathmin and SCG10, overexpressed SCLIP, RB3 and RB3″ were able to depolymerize interphase microtubules. Altogether, our results demonstrate in vivo the functional conservation of the stathmin domain within each protein of the stathmin family, with a microtubule destabilizing activity most likely essential for their specific biological function(s).

scholarly journals A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 424-434 ◽  
Author(s):  
J G Izant ◽  
J A Weatherbee ◽  
J R McIntosh
Keyword(s):  
Mitotic Spindle ◽  
Microtubule Network ◽  
Mitotic Apparatus ◽  
Microtubule Associated Proteins ◽  
Immunoglobulin Preparations ◽  
Rapid Dissolution ◽  
Associated Proteins ◽  
Cultured Human Cells

Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network.

Abstract 378: Macrophage ß3 Integrin is an Important Player in Inflammation and Cellular Plasticity

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Cristina A Wolf ◽  
Izabela Bobak ◽  
Xinming Su ◽  
Ellen Damm ◽  
Katherine N Weilbaecher ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Cellular Response ◽  
Foam Cell ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Cellular Plasticity ◽  
Β3 Integrin ◽  
Anti Inflammatory

Communication between cells and the surrounding environment is a crucial mechanism for survival. Integrins are membrane-bound molecules that are involved in signaling between the cells and the extracellular matrix, thereby influencing cytoskeletal stability and intracellular signaling. β3 integrin and its binding partner αv form the αvβ3 heterodimer that is expressed in various cells. We and others have described the consequences of its absence in inflammation, atherosclerosis and cancer in vivo. However, the distinct role of this integrin as a signaling molecule and the consequences of its absence for macrophage structure remain mostly elusive. Our aim is to further characterize the phenotype of β3-deficient (β3-/-) bone marrow-derived macrophages (BMDM) under stimulatory conditions (LPS and LDLs) compared to control cells in vitro. qPCR, WB, ELISA, migration, proliferation assays were used to investigate β3-/- BMDM and controls (wt BMDM and Raw 264.7). LPS was described to be not only pro- but also anti-inflammatory in a time-dependent manner. We show that LPS stimulation leads to high expression of pro-inflammatory cytokines (IL-1β and TNFα) shortly after treatment, while expression of anti-inflammatory cytokine (IL-10) arises at a later stage (12h post stimulation). Interestingly, β3-/- BMDM express more IL-1β than controls. IL-10 expression appears much earlier in β3-/- BMDM (6h post stimulation) but is reduced after 12h, indicating a faster and higher cellular response in the absence of the β3 integrin. OxLDL, the leading cause to foam cell formation, stimulates the expression of IL-1β in controls and β3-/- BMDM with the latter expressing significantly less of this cytokine indicating that lack of β3 causes differential cellular responses after LPS and oxLDL stimulation. Other LDL forms tested (nLDL, acLDL, cLDL) did not have any effect on IL-1β expression. In addition, we identified a higher proliferation rate in the β3-/- BMDM when cultured with M-CSF and a migration deficit in response to LPS, M-CSF and VEGF. Taken together, our results show that macrophage β3 deficiency causes differential cellular plasticity depending on the stimulus, with functional consequences that could be essential in inflammation and atherosclerosis.

scholarly journals Characterization of proteins immunologically related to brain microtubule-associated protein MAP-1B in non-neural cells

1989 ◽  
Vol 92 (4) ◽  
pp. 607-620
Author(s):  
J. Diaz-Nido ◽  
J. Avila
Keyword(s):  
Mitotic Spindle ◽  
Polyclonal Antibodies ◽  
Microtubule Assembly ◽  
Neural Cells ◽  
Microtubule Network ◽  
Cytoplasmic Microtubule ◽  
Protein Map ◽  
Dividing Cells

Brain microtubule-associated protein MAP-1 is composed of at least two polypeptides, MAP-1A and MAP-1B, which are among the main components of the neural cytoskeleton. Specific monoclonal and polyclonal antibodies against MAP-1B stain nuclei, mitotic spindles, centrosomes and the cytoplasmic microtubule network of different non-neural cells studied by immunofluorescence microscopy. It appears that these cells contain two proteins of 325K and 220K (K = 10(3) Mr), which are immunologically related to brain MAP-1B. The 325K protein, which is localized to the cytoplasmic microtubule network, the centrosome and the mitotic spindle, seems to be structurally related to the neural MAP-1B, as judged from their similar peptide maps and phosphorylation patterns. The 220K protein, which is localized to the nuclear matrix in interphase cells and to the mitotic spindle in dividing cells, has a proteolytic profile different from that of neural MAP-1B and is phosphorylated to a much lesser extent than the 325K protein. Both proteins bind tubulin in vitro, which suggests that they may participate in microtubule assembly in vivo; the 325K protein could perform such a role during the entire cell cycle, while the 220K protein could be implicated in the formation of the mitotic spindle.

scholarly journals Therapeutic potential of a novel prodrug of green tea extract in induction of apoptosis via ERK/JNK and Akt signaling pathway in human endometrial cancer

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Gene Chi Wai Man ◽  
Jianzhang Wang ◽  
Yi Song ◽  
Jack Ho Wong ◽  
Yu Zhao ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Green Tea ◽  
Intracellular Signaling ◽  
Low Dose ◽  
Expression Profiles ◽  
Dependent Manner ◽  
Green Tea Polyphenol ◽  
Ec Cells

Abstract Background Previous studies have shown a major green tea polyphenol (−)-epigallocatechin-3-gallate ((−)-EGCG) as a powerful anti-cancer agent. However, its poor bioavailability and requirement of a high dosage to manifest activity have restricted its clinical application. Recently, our team synthesized a peracetate-protected derivative of EGCG, which can act as a prodrug of (−)-EGCG (ProEGCG) with enhanced stability and improved bioavailability in vitro and in vivo. Herein, we tested the therapeutic efficacy of this novel ProEGCG, in comparison to EGCG, toward human endometrial cancer (EC). Methods In this study, the effects of ProEGCG and EGCG treatments on cell growth, cell survival and modulation of intracellular signaling pathways in RL95–2 and AN3 CA EC cells were compared. The antiproliferative effect was evaluated by cell viability assay. Apoptosis was measured by annexin/propidium iodide staining. Expression of mitogen-activated protein kinases, markers of proliferation and apoptosis were measured by immunoblot analysis. In addition, the effects of ProEGCG and EGCG on tumor growth, vessel formation and gene expression profiles on xenograft models of the EC cells were investigated. Results We found that treatment with ProEGCG, but not EGCG, inhibited, in a time- and dose-dependent manner, the proliferation and increased apoptosis of EC cells. Treatment with low-dose ProEGCG significantly enhanced phosphorylation of JNK and p38 MAPK and inhibited phosphorylation of Akt and ERK which are critical mediators of apoptosis. ProEGCG, but not EGCG, elicited a significant decrease in the growth of the EC xenografts, promoted apoptotic activity of tumour cells in the EC xenografts, and decreased microvessel formation, by differentially suppressing anti-apoptotic molecules, NOD1 and NAIP. Notably, no obvious adverse effects were detected. Conclusions Taken together, ProEGCG at a low dose exhibited anticancer activity in EC cells through its anti-proliferative, pro-apoptotic and anti-tumor actions on endometrial cancer in vitro and in vivo. In contrast, a low dose of EGCG did not bring about similar effects. Importantly, our data demonstrated the efficacy and safety of ProEGCG which manifests the potential of a novel anticancer agent for the management of endometrial cancer.

scholarly journals Identification of the In Vivo Casein Kinase II Phosphorylation Site within the Homeodomain of the Cardiac Tisue-Specifying Homeobox Gene Product Csx/Nkx2.5

1999 ◽  
Vol 19 (1) ◽  
pp. 526-536 ◽  
Author(s):  
Hideko Kasahara ◽  
Seigo Izumo
Keyword(s):  
Mutational Analysis ◽  
Intracellular Localization ◽  
Phosphorylation Site ◽  
Homeobox Gene ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Site Directed Mutagenesis ◽  
Phosphopeptide Mapping

ABSTRACT Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2.5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes.

scholarly journals Peptide Uptake Is Essential forBorrelia burgdorferiViability and Involves Structural and Regulatory Complexity of its Oligopeptide Transporter

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Ashley M. Groshong ◽  
Abhishek Dey ◽  
Irina Bezsonova ◽  
Melissa J. Caimano ◽  
Justin D. Radolf
Keyword(s):  
Amino Acid ◽  
Borrelia Burgdorferi ◽  
Intracellular Signaling ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Wide Range ◽  
Peptide Uptake ◽  
Oligopeptide Transport

ABSTRACTBorrelia burgdorferiis an extreme amino acid (AA) auxotroph whose genome encodes few free AA transporters and an elaborate oligopeptide transport system (B. burgdorferiOpp [BbOpp]).BbOpp consists of five oligopeptide-binding proteins (OBPs), two heterodimeric permeases, and a heterodimeric nucleotide-binding domain (NBD). Homology modeling based on the crystal structure of ligandedBbOppA4 revealed that each OBP likely binds a distinct range of peptides. Transcriptional analyses demonstrated that the OBPs are differentially and independently regulated whereas the permeases and NBDs are constitutively expressed. A conditional NBD mutant failed to divide in the absence of inducer and replicated in an IPTG (isopropyl-β-d-thiogalactopyranoside) concentration-dependent manner. NBD mutants grown without IPTG exhibited an elongated morphotype lacking division septa, often with flattening at the cell center due to the absence of flagellar filaments. Following cultivation in dialysis membrane chambers, NBD mutants recovered from rats not receiving IPTG also displayed an elongated morphotype. The NBD mutant was avirulent by needle inoculation, but infectivity was partially restored by oral administration of IPTG to infected mice. We conclude that peptides are a major source of AAs forB. burgdorferibothin vitroandin vivoand that peptide uptake is essential for regulation of morphogenesis, cell division, and virulence.IMPORTANCEBorrelia burgdorferi, the causative agent of Lyme disease, is an extreme amino acid (AA) auxotroph with a limited repertoire of annotated single-AA transporters. A major issue is how the spirochete meets its AA requirements as it transits between its arthropod vector and mammalian reservoir. While previous studies have confirmed that theB. burgdorferioligopeptide transport (opp) system is capable of importing peptides, the importance of the system for viability and pathogenesis has not been established. Here, we evaluated theoppsystem structurally and transcriptionally to elucidate its ability to import a wide range of peptides during the spirochete’s enzootic cycle. Additionally, using a novel mutagenesis strategy to abrogateopptransporter function, we demonstrated that peptide uptake is essential for bacterial viability, morphogenesis, and infectivity. Our studies revealed a novel link between borrelial physiology and virulence and suggest that peptide uptake serves an intracellular signaling function regulating morphogenesis and division.

scholarly journals Peptidyl-Prolyl Isomerase 1 (Pin1) Serves as a Coactivator of Steroid Receptor by Regulating the Activity of Phosphorylated Steroid Receptor Coactivator 3 (SRC-3/AIB1)

2005 ◽  
Vol 25 (21) ◽  
pp. 9687-9699 ◽  
Author(s):  
Ping Yi ◽  
Ray-Chang Wu ◽  
Joshua Sandquist ◽  
Jiemin Wong ◽  
Sophia Y. Tsai ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Intracellular Signaling ◽  
Steroid Receptors ◽  
Steroid Receptor ◽  
Dependent Manner ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Steroid Receptor Coactivator

ABSTRACT Steroid receptor coactivator 3 (SRC-3/AIB1) interacts with steroid receptors in a ligand-dependent manner to activate receptor-mediated transcription. A number of intracellular signaling pathways initiated by growth factors and hormones induce phosphorylation of SRC-3, regulating its function and contributing to its oncogenic potential. However, the range of mechanisms by which phosphorylation affects coactivator function remains largely undefined. We demonstrate here that peptidyl-prolyl isomerase 1 (Pin1), which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, interacts selectively with phosphorylated SRC-3. In addition, Pin1 and SRC-3 activate nuclear-receptor-regulated transcription synergistically. Depletion of Pin1 by small interfering RNA (siRNA) reduces hormone-dependent transcription from both transfected reporters and an endogenous steroid receptor target gene. We present evidence that Pin1 modulates interactions between SRC-3 and CBP/p300. The interaction is enhanced in vitro and in vivo by Pin1 and diminished when cellular Pin1 is reduced by siRNA or in stable Pin1-depleted cell lines. Depletion of Pin1 in MCF-7 human breast cancer cells reduces the endogenous estrogen-dependent recruitment of p300 to the promoters of estrogen receptor-dependent genes. Pin1 overexpression enhanced SRC-3 cellular turnover, and depletion of Pin1 stabilized SRC-3. Our results suggest that Pin1 functions as a transcriptional coactivator of nuclear receptors by modulating SRC-3 coactivator protein-protein complex formation and ultimately by also promoting the turnover of the activated SRC-3 oncoprotein.

Truncated Cell-Surface CD19 As a Conditional Suicide Switch For Adoptive T Cell Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1660-1660 ◽  
Author(s):  
Lihua Elizabeth Budde ◽  
Armen Mardiros ◽  
Wen-Chung Chang ◽  
Xiuli Wang ◽  
Carolina Berger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Intracellular Signaling ◽  
Flow Cytometric Analysis ◽  
Dependent Manner ◽  
Cell Immunotherapy ◽  
T Cell Immunotherapy

Background Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe Methods for removing transferred cells after treatment an important consideration. In addition, there is a lack of effective commercially available agents which allow for monitoring of CAR expression, tracking, isolating, and eliminating CAR- transduced cells. Therefore, adoptive T cell immunotherapy would benefit from a molecule which is stably expressed on the cell surface, of human origin, easily detected on transduced cells, lacking active biological function at baseline and capable of effectively ablating transduced cells on demand. Truncated CD19 (CD19t) harbors excellent features to be such a molecule. Its truncation shortens the intracytoplasmic domain to only 19 amino acids with removal of all conserved tyrosine residues that mediate known intracellular signaling transduction. It has been used successfully to mark transduced CAR T cells by several research groups. In this study, we set out to evaluate the activity of this truncated CD19 as a conditional suicide switch. Methods Lentiviral constructs containing a CD20 CAR and CD19t were used to transduce Jurkat T cells and primary human T cells to generate cells that express both molecules on the cell surface. CD19-mediated selection was carried out using PE conjugated anti-CD19 antibody. Internalization experiments were performed using transduced Jurkat cells that were kept at 4°C or 37°C. Surface CD19 expression was determined by flow cytometric analysis at 0 hour, 1 hour, 2 hours, and 4 hours after initial primary anti-CD19 antibody staining. NIH3T3 cells with truncated CD19 expressed on the surface (NIH3T3-19t) were generated and used for in vitro ablation experiments. Cells were left untreated or incubated withincreasing concentrations of CD19-ETA’, an anti-CD19 Pseudomonas toxin conjugate. The viability of NIH3T3-CD19t was determined by trypan blue exclusion at various time points. Results Using flow cytometry, we confirmed the expression of CAR and truncated CD19 on the transduced cell surface. Truncated CD19 was able to enrich transduced cells to more than 90% purity when used as a selectable marker. For CD19t to function as a conditional suicide switch, it needs to retain its ability to mediate antigen-antibody conjugate internalization. As expected, only up to 10% of CD19t remained on the surface of transduced cells after 4-hour period at 37°C. On the contrary, surface CD19t expression level remained largely unchanged when the cells were incubated at 4°C for 4 hours. CD19t also mediated robust ablation of transduced cells in vitro. More than 90% of transduced cells were ablated after 72-hour incubation with 10ug/ml CD19-ETA’. Mouse xenograft experiments are currently ongoing to test the in vivo tracking ability of CD19t and its ablation effect of transferred T cells upon administration of anti-CD19-drug conjugates. Conclusions These in vitro Results suggest that CD19t retains the ability to mediate antibody internalization upon its engagement. When exposed to an anti-CD19-drug conjugate, cells expressing truncated CD19 are effectively ablated in a dose dependent manner. We therefore predict that CD19t will be an excellent molecule to mark, select, track and eliminate modified T cells in vivo and it will be a useful tool for detection of engineered T cells and improvement of the safety of adoptive T cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals LMTK1 regulates dendritic formation by regulating movement of Rab11A-positive endosomes

2014 ◽  
Vol 25 (11) ◽  
pp. 1755-1768 ◽  
Author(s):  
Tetsuya Takano ◽  
Tomoki Urushibara ◽  
Nozomu Yoshioka ◽  
Taro Saito ◽  
Mitsunori Fukuda ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Phosphorylation Site ◽  
Dendrite Growth ◽  
Endosomal Trafficking ◽  
Dependent Manner ◽  
Dendrite Development ◽  
Membrane Components ◽  
And Function

Neurons extend two types of neurites—axons and dendrites—that differ in structure and function. Although it is well understood that the cytoskeleton plays a pivotal role in neurite differentiation and extension, the mechanisms by which membrane components are supplied to growing axons or dendrites is largely unknown. We previously reported that the membrane supply to axons is regulated by lemur kinase 1 (LMTK1) through Rab11A-positive endosomes. Here we investigate the role of LMTK1 in dendrite formation. Down-regulation of LMTK1 increases dendrite growth and branching of cerebral cortical neurons in vitro and in vivo. LMTK1 knockout significantly enhances the prevalence, velocity, and run length of anterograde movement of Rab11A-positive endosomes to levels similar to those expressing constitutively active Rab11A-Q70L. Rab11A-positive endosome dynamics also increases in the cell body and growth cone of LMTK1-deficient neurons. Moreover, a nonphosphorylatable LMTK1 mutant (Ser34Ala, a Cdk5 phosphorylation site) dramatically promotes dendrite growth. Thus LMTK1 negatively controls dendritic formation by regulating Rab11A-positive endosomal trafficking in a Cdk5-dependent manner, indicating the Cdk5-LMTK1-Rab11A pathway as a regulatory mechanism of dendrite development as well as axon outgrowth.

Two splice variants of Golgi-microtubule-associated protein of 210 kDa (GMAP-210) differ in their binding to the cis-Golgi network

2001 ◽  
Vol 357 (3) ◽  
pp. 699-708 ◽  
Author(s):  
Francisco RAMOS-MORALES ◽  
Carmen VIME ◽  
Michel BORNENS ◽  
Concepción FEDRIANI ◽  
Rosa M. RIOS
Keyword(s):  
Golgi Apparatus ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Genomic Sequence ◽  
Splice Variants ◽  
Microtubule Network ◽  
Terminal Domain ◽  
Golgi Network

GMAP-210 (Golgi-microtubule-associated protein of 210kDa) is a peripheral Golgi protein that interacts with the minus end of microtubules through its C-terminus and with cis-Golgi network membranes through its N-terminus; it participates in the maintenance of the structural integrity of the Golgi apparatus [Infante, Ramos-Morales, Fedriani, Bornens and Rios (1999) J. Cell Biol. 145, 83–98]. We report here the cloning of a new isoform of GMAP-210 that lacks amino acid residues 105–196. On the basis of the analysis of the gmap-210 genomic sequence, we propose that the small isoform, GMAP-200, arises from alternative splicing of exon 4 of the primary transcript. Overexpression of GMAP-200 induces perturbations in both the Golgi apparatus and the microtubule network that are similar to those previously reported for GMAP-210 overexpression. We show that both isoforms are able to oligomerize under overexpression conditions. Analysis in vitro and in vivo, with the green fluorescent protein as a marker, reveals that the binding of the N-terminal domain of GMAP-200 to the cis-Golgi network membranes is lower than that of the N-terminal domain of GMAP-210. Implications for the regulation of interaction between the cis-Golgi network and microtubules are discussed.

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