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1.
Oncogene ; 32(6): 768-76, 2013 Feb 07.
Article in English | MEDLINE | ID: mdl-22430209

ABSTRACT

Although activating mutations of PIK3CA are frequent in urothelial carcinoma (UC), no information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum, which has a large excess of helical domain mutations. We investigated the phenotypic and signaling consequences of hotspot and UC-specific rare PIK3CA mutations in immortalized normal human urothelial cells (NHUC) and mouse fibroblasts (NIH3T3). Our results indicate that in NHUC, rare mutant forms and all three hotspot mutant forms of PIK3CA can activate the PI3K/AKT pathway. The relative frequency at which helical domain and kinase domain mutations are found in UC is related to their potency in inducing signaling downstream of AKT and to the phenotypic effects induced in this cell type (E545K>E542K>H1047R). Helical domain mutations E542K and E545K conferred a significant proliferative advantage at confluence and under conditions of nutrient depletion, and increased cellular resistance to anoikis. Both helical and kinase domain mutants induced increased NHUC cell motility and migration towards a chemoattractant, though no significant differences were found between the mutant forms. In NIH3T3 cells, the kinase domain mutant H1047R induced high levels of AKT activation, but helical domain mutants were significantly less potent and this was reflected in their relative abilities to confer anchorage-independent growth. Our findings indicate that the effects of mutant PIK3CA are both cell type- and mutation-specific. Helical domain mutations in PIK3CA may confer a selective advantage in the urothelium in vivo by overcoming normal contact-mediated inhibitory signals and allowing proliferation in nutrient-limiting conditions. Mutant forms of PIK3CA may also stimulate intraepithelial cell movement, which could contribute to spread of cells within the urothelium.


Subject(s)
Phosphatidylinositol 3-Kinases/genetics , Signal Transduction , Urinary Bladder Neoplasms/genetics , Urothelium/metabolism , Animals , Class I Phosphatidylinositol 3-Kinases , Fibroblasts/metabolism , Humans , Mice , Mutation , NIH 3T3 Cells , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism
2.
Oncogene ; 29(1): 150-5, 2010 Jan 07.
Article in English | MEDLINE | ID: mdl-19802009

ABSTRACT

The phosphatidylinositol-3-kinase (PI3 kinase)-AKT pathway is frequently activated in cancer. Recent reports have identified a transforming mutation of AKT1 in breast, colorectal, ovarian and lung cancers. We report here the occurrence of this mutation in bladder tumours. The AKT1 G49A (E17K) mutation was found in 2/44 (4.8%) bladder cancer cell lines and 5/184 (2.7%) bladder tumours. Cell lines expressing mutant AKT1 show constitutive AKT1 activation under conditions of growth factor withdrawal. We also detected a novel AKT1 mutation G145A (E49K). This mutation also enhances AKT activation and shows transforming activity in NIH3T3 cells, though activity is weaker than that of E17K. Enhanced activation of AKT1 when E17K and E49K mutations are in tandem suggests that they can co-operate.


Subject(s)
Mutation, Missense , Proto-Oncogene Proteins c-akt/genetics , Urinary Bladder Neoplasms/genetics , Animals , Base Sequence , Blotting, Western , Cell Line, Tumor , Cells, Cultured , DNA Mutational Analysis , Enzyme Activation/genetics , Gene Frequency , Humans , Mice , NIH 3T3 Cells , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Serine/metabolism , Threonine/metabolism , Transfection , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology
3.
Oncogene ; 27(17): 2494-500, 2008 Apr 10.
Article in English | MEDLINE | ID: mdl-17968321

ABSTRACT

The microtubule (MT)-associated protein EB1 localizes to and promotes growth at MT plus ends. The MT depolymerizing kinesin MCAK has also been reported to track growing MT plus ends. Here, we confirm that human MCAK colocalizes with EB1 at growing MT ends when expressed as a GFP fusion protein in transfected cells. We show that MCAK associates with the C-terminus of EB1 and EB3 but much less efficiently with RP1. EB1 associates with the N-terminal localization and regulatory domain in MCAK but not with the motor domain of the protein. The interaction is competitive with the binding of other EB1 ligands and does not require MTs. Knockdown of EB1 expression using siRNA impaired the ability of GFP-MCAK to localize to MT tips in transfected cells. We propose that MCAK is targeted to growing MT ends by EB1, that MCAK is held in an inactive conformation when associated with EB1 and that this could provide the basis for a mechanism that facilitates rapid switching between phases of MT growth and depolymerization.


Subject(s)
Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Animals , Cell Line , Chlorocebus aethiops , Humans , Kinesins/genetics , Ligands , Microtubule-Associated Proteins/genetics , Protein Binding
4.
Cell Motil Cytoskeleton ; 63(8): 483-92, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16767746

ABSTRACT

The adenomatous polyposis coli (APC) tumor suppressor protein is mutated in most colorectal carcinomas. In addition to its role in WNT signaling it is proposed to be involved in both cell migration and mitosis. Although a variety of studies have shown an APC localization along lateral membranes of adjacent epithelial cells the existence of a cortical APC localization in mammalian cells remains controversial. To address this we have used matched rat epithelial (NRK-52E) and fibroblast (NRK-49F) cell lines to investigate the localization of APC. Subconfluent cultures of NRK-52E and -49F cells displayed microtubule-associated APC populations by immunostaining. However, confluent NRK-52E, but not -49F monolayers, exhibited a cortical APC distribution. Cortical APC localized in close proximity to a number of cell junction proteins in a microtubule-independent manner while calcium switch experiments suggested that APC was recruited to the cortex only when junction assembly was complete. Confluent NRK-49F and -52E cells also showed contrasting APC localizations in response to monolayer wounding. Our data suggests APC cortical localization is a feature of confluent epithelioid cells and that the subcellular distribution of APC is therefore dependent upon both cell type and context.


Subject(s)
Adenomatous Polyposis Coli Protein/metabolism , Epithelial Cells/metabolism , Fibroblasts/metabolism , Alternative Splicing , Animals , Cell Adhesion/physiology , Cell Line , Epithelial Cells/cytology , Fibroblasts/cytology , Microtubules/metabolism , Protein Isoforms , Rats , Spindle Apparatus/metabolism
5.
Mol Biol Cell ; 13(10): 3627-45, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12388762

ABSTRACT

EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150(Glued) was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.


Subject(s)
Centrosome/metabolism , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Recombinant Fusion Proteins/metabolism , Adenomatous Polyposis Coli Protein/genetics , Adenomatous Polyposis Coli Protein/metabolism , Animals , Antibodies, Monoclonal/metabolism , Antineoplastic Agents/metabolism , Cell Line , Cytoskeleton/metabolism , Dynactin Complex , Dyneins/metabolism , Epitope Mapping , Golgi Apparatus/metabolism , Humans , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Neoplasm Proteins , Nocodazole/metabolism , Protein Binding , Recombinant Fusion Proteins/genetics , Tubulin/metabolism
6.
Brain Res Mol Brain Res ; 98(1-2): 145-52, 2002 Jan 31.
Article in English | MEDLINE | ID: mdl-11834307

ABSTRACT

EB1 is a microtubule associated protein which interacts with the APC tumour suppressor protein and components of the cytoplasmic dynein/dynactin complex. EB1 is also a specific marker of growing microtubule tips. Here we demonstrate that EB1 protein levels are increased during axon but not dendrite formation in differentiated N2A neuroblastoma cells, and that EB1 localises to microtubule tips throughout extending neurites in these cells. In N2A axons, analysis of the ratio of EB1/beta-tubulin fluorescence demonstrated that the distal tip region contained the highest proportion of polymerising microtubules. Time-lapse confocal imaging of an EB1-GFP fusion protein in transfected N2A cells directly revealed the dynamics of microtubule extension in neurites, and demonstrated the existence of unusual, discrete knots of microtubule polymerisation at the periphery of non-process bearing cells which may represent an early event in neurite outgrowth. We conclude that EB1 localisation can be used to identify and analyse sites of microtubule polymerisation at a high resolution during neurite development, a process to which it may contribute.


Subject(s)
Axons/metabolism , Microtubule-Associated Proteins/analysis , Microtubules/metabolism , Nerve Tissue Proteins/analysis , Animals , Axons/ultrastructure , Biopolymers , Dendrites/chemistry , Dendrites/ultrastructure , Green Fluorescent Proteins , Luminescent Proteins/analysis , Luminescent Proteins/genetics , Macromolecular Substances , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Microtubules/chemistry , Nerve Tissue Proteins/genetics , Neuroblastoma/pathology , Protein Interaction Mapping , Recombinant Fusion Proteins/analysis , Tubulin/analysis , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/ultrastructure
7.
Eur J Cell Biol ; 80(12): 749-53, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11831388

ABSTRACT

Spindle positioning in animal cells is thought to rely upon the interaction of astral microtubules with the cell cortex. Information on the dynamics of astral microtubules during this process is scarce, in part because of the difficulty in visualising these microtubules by light microscopy. EB1 is a protein which specifically localises to growing microtubule distal tips. Immunostaining for EB1 therefore represents a powerful method for visualising the distribution of growing microtubule tips within cells. In this study we used EB1 immunostaining in mitotic NRK-52E cells to quantitatively analyse the length and number of growing astral microtubules during metaphase and anaphase. We observed a dramatic increase in growing astral microtubule length and number during anaphase. Furthermore, drug treatments which specifically destroyed astral microtubules resulted in an increase in misaligned anaphase but not metaphase spindles. We suggest that an anaphase-specific increase in growing astral microtubule length and number facilitates the maintenance of a correctly aligned spindle in mitotic NRK-52E cells.


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Anaphase , Animals , Cell Line , Fluorescent Antibody Technique , Microtubule-Associated Proteins/analysis , Microtubules/chemistry , Rats , Spindle Apparatus/metabolism
8.
Oncogene ; 19(15): 1950-8, 2000 Apr 06.
Article in English | MEDLINE | ID: mdl-10773885

ABSTRACT

The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.


Subject(s)
Cytoskeletal Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Adenomatous Polyposis Coli Protein , Amino Acid Sequence , Humans , Microtubules/metabolism , Mitosis , Molecular Sequence Data , Neoplasm Proteins/metabolism , Phosphorylation , Tumor Cells, Cultured
9.
Eur J Cancer ; 36(4): 525-32, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10717531

ABSTRACT

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.


Subject(s)
Colorectal Neoplasms/genetics , Cytoskeletal Proteins/metabolism , Genes, APC/genetics , Mutation/genetics , Neoplasm Proteins/genetics , Adenomatous Polyposis Coli Protein , Colorectal Neoplasms/metabolism , Cytoskeletal Proteins/genetics , Genetic Vectors , Humans , Mutagenesis, Site-Directed , Neoplasm Proteins/metabolism , Phosphorylation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tyrosine/metabolism
10.
Oncogene ; 17(26): 3471-7, 1998 Dec 31.
Article in English | MEDLINE | ID: mdl-10030671

ABSTRACT

The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.


Subject(s)
Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , DNA-Binding Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Adenomatous Polyposis Coli Protein , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , COS Cells/drug effects , COS Cells/metabolism , Cell Cycle/physiology , Cells, Cultured , Cricetinae , Cytoskeletal Proteins/genetics , DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , Fluorescent Antibody Technique , Humans , Microtubule-Associated Proteins/analysis , Microtubule-Associated Proteins/genetics , Microtubules/drug effects , Microtubules/metabolism , Nocodazole/pharmacology , Paclitaxel/pharmacology
11.
Neuroscience ; 81(2): 553-63, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9300441

ABSTRACT

The adenomatous polyposis coli tumour suppressor protein is highly expressed in developing rodent brain, but its function is unclear. Recent studies have suggested a role for this protein in regulating microtubule dynamics. Neuro 2A mouse neuroblastoma cells were previously thought not to express this protein. Using immunochemical techniques, this report corrects this observation. Immunoreactive bands of a size consistent with that of the full-length protein were observed by western blotting. Using immunocytochemistry, punctate immunoreactivity localized to areas of the cell containing microtubules, particularly neurite growth cones, in a distribution suggesting a role in neuritogenesis and growth cone extension. The protein did not localize to actin-rich cellular structures, and perturbation of the actin cytoskeleton had no effect upon this distribution. Treatment of cells with taxol to stabilize microtubules caused the concentration of the immunoreactive puncta to the tips of microtubules and areas along the axis of potential microtubule assembly. Treatment of cells with the microtubule disrupting reagent nocodazole showed that over shorter times the punctate distribution was not dependent upon polymerized microtubules. However, at longer incubation times a decrease in punctate immunostaining was observed. These results indicate that the intracellular distribution of the adenomatous polyposis coli protein is dependent upon microtubule but not actin dynamics. A role for this protein in the regulation of directed microtubule assembly is suggested.


Subject(s)
Adenomatous Polyposis Coli/metabolism , Microtubules/physiology , Neuroblastoma/metabolism , Animals , Antibodies/immunology , Immunohistochemistry , Mice , Tumor Cells, Cultured
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