Identification of centrosomal proteins in a human lymphoblastic cell line.

Gosti-Testu, Françoise; Marty, Marie-Chantal; Bergès, Josette; Maunoury, R; Bornens, Michel

doi:10.1002/j.1460-2075.1986.tb04533.x

Cited by 71 publications

(39 citation statements)

References 45 publications

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“…All RACE products were cloned into pBluescript-T (21). Further human sequence information was obtained from the total sequencing of an expressed sequence-tagged cDNA from the human promyeloblast cell line KG1a, PMY2245 (22). Finally, biotinylated recombinant R II was used to screen a human tracheal cDNA library in LambdaZap using a standard protein overlay technique.…”

Section: Methodsmentioning

confidence: 99%

“…The characteristics of the 350-kDa AKAP that we have cloned appear similar to those of the centrosomal AKAP350 identified by Keryer et al (9) in an R II blot overlay of isolated centrosome proteins from the KE37 human lymphoblast cell line. These investigators immunoprecipitated the protein using the serum 0013 polyclonal antibody, previously reported as a specific marker for human centrosomes (20,22,38). No further characterization of this protein in the literature has been submitted, however, since the initial report.…”

Section: Fig 8 Distribution Of Akap350mentioning

confidence: 99%

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AKAP350, a Multiply Spliced Protein Kinase A-anchoring Protein Associated with Centrosomes

Schmidt¹,

Dransfield²,

Claudio³

et al. 1999

Journal of Biological Chemistry

133

152

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Protein kinase A-anchoring proteins (AKAPs) localize the second messenger response to particular subcellular domains by sequestration of the type II protein kinase A. Previously, AKAP120 was identified from a rabbit gastric parietal cell cDNA library; however, a monoclonal antibody raised against AKAP120 labeled a 350-kDa band in Western blots of parietal cell cytosol. Recloning has now revealed that AKAP120 is a segment of a larger protein, AKAP350. We have now obtained a complete sequence of human gastric AKAP350 as well as partial cDNA sequences from human lung and rabbit parietal cells. The genomic region containing AKAP350 is found on chromosome 7q21 and is multiply spliced, producing at least three distinct AKAP350 isoforms as well as yotiao, a protein associated with the N-methyl-D-aspartate receptor. Rabbit parietal cell AKAP350 is missing a sequence corresponding to a single exon in the middle of the molecule located just after the yotiao homology region. Two carboxyl-terminal splice variants were also identified. Both of the major splice variants showed tissue-and cell-specific expression patterns. Immunofluorescence microscopy demonstrated that AKAP350 was associated with centrosomes in many cell types. In polarized Madin-Darby canine kidney cells, AKAP350 localized asymmetrically to one pole of the centrosome, and nocodazole did not alter its localization. During the cell cycle, AKAP350 was associated with the centrosomes as well as with the cleavage furrow during anaphase and telophase. Several epithelial cell types also demonstrated noncentrosomal pools of AKAP350, especially parietal cells, which contained multiple cytosolic immunoreactive foci throughout the cells. The localization of AKAP350 suggests that it may regulate centrosomal and noncentrosomal cytoskeletal systems in many different cell types.Transduction of signals from extracellular stimuli is most commonly accomplished via ligand-receptor binding and generation of a second messenger response. While increases in intracellular second messengers have traditionally been viewed as global cellular events, second messenger effects are often limited to particular regions or organelles within cells. Investigations over the past decade have led to a greater understanding of the mechanisms responsible for the compartmentalization of second messenger effects. These studies have identified a diverse group of scaffolding proteins that sequester both protein kinases and protein phosphatases within specific cellular domains (1, 2). In the case of cAMP-dependent protein kinases, protein kinase A-anchoring proteins (AKAPs) 1 tether the protein kinase A holoenzyme through binding to the regulatory subunit dimer. A growing group of AKAPs that bind the regulatory subunit of type II protein kinase A (R II ) have been reported over the past several years. The first R II -binding protein was identified over 15 years ago when microtubule-associated protein 2 (MAP-2) was described (3, 4). Since that time, several AKAPs have been identified, localizing the type...

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Section: Methodsmentioning

confidence: 99%

Section: Fig 8 Distribution Of Akap350mentioning

confidence: 99%

AKAP350, a Multiply Spliced Protein Kinase A-anchoring Protein Associated with Centrosomes

Schmidt¹,

Dransfield²,

Claudio³

et al. 1999

Journal of Biological Chemistry

133

152

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“…Mitotic centrosomes were purified from HeLa S3 cells arrested in mitosis using 10 M S-trityl-l-cysteine using the Bornens protocol (Gosti-Testu et al, 1986). Mitotic centrosomes fractionate differently than the centrosomes normally purified from asynchronous populations of cells so care must be taken to assay each fraction for centrosomes enrichment.…”

Section: Preparation Of Other Reaction Componentsmentioning

confidence: 99%

Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

Chang

Coughlin

Mitchison

2009

MBoC

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Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.

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“…An increase in centrosome-specific staining with anticentrosome antibodies occurs during G2 and a dramatic decrease takes place at the onset of anaphase (Gosti-Testu et al, 1986). These centrosomal modifications, which are likely to correspond to a rise and fall of microtubule nucleating activity (for a review, see Bornens and Karsenti, 1984), may well involve changes in phosphorylation.…”

Section: Introductionmentioning

confidence: 99%

p34cdc2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase.

et al. 1989

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The cdc2+ gene product p34cdc2 is located immunocytochemically in both the nucleus and cytoplasm of human cells. It is uniformly distributed throughout the cytoplasm and is irregularly distributed in the nucleus. Part of p34cdc2 is associated with the centrosome and centrosomal staining increases late in the cell cycle and at the onset of mitosis. This distribution is corroborated by cell fractionation which also indicates that slower migrating forms of p34cdc2 are found in isolated centrosomes and in Triton-insoluble fractions. We propose that one role of the p34Cdc2 protein kinase is to modify the centrosome bringing about formation of the mitotic spindle. At anaphase p34cdc2 becomes associated with vesicles in the middle of the cell between the reforming nuclei. A similar location is found for pl3SUCl and we suggest that the vesicular localization plays a role in p34cdc2 kinase inactivation at the end of mitosis.

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Identification of centrosomal proteins in a human lymphoblastic cell line.

Cited by 71 publications

References 45 publications

AKAP350, a Multiply Spliced Protein Kinase A-anchoring Protein Associated with Centrosomes

AKAP350, a Multiply Spliced Protein Kinase A-anchoring Protein Associated with Centrosomes

Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly

p34cdc2 is located in both nucleus and cytoplasm; part is centrosomally associated at G2/M and enters vesicles at anaphase.

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