Identification of novel M phase phosphoproteins by expression cloning.

Matsumoto-Taniura, N; Pirollet, Fabienne; Monroe, Robin S.; Gerace, Larry; Westendorf, Joanne M.

doi:10.1091/mbc.7.9.1455

Cited by 175 publications

(185 citation statements)

References 44 publications

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“…Notably, the transcription factor Oct-1 is highly phosphorylated during mitosis, resulting in translocation from the chromosome to the cytosol (41). In addition, many other mitotic phosphoproteins exhibit a localization pattern similar to that observed for the G-97 PP-1 antibody (42,43).…”

Section: Discussionmentioning

confidence: 65%

Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase

Kwon

Lee

Choi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

204

192

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Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression. In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity. To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C␣ phosphorylated at T320. Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 was reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1͞PP-2A inhibitor, calyculin A. Immunof luorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase. Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions. Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction. These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.Protein phosphorylation is widely recognized as the major mechanism that controls cell cycle progression. A family of cyclin-dependent protein kinases (CDKs) have been identified and found to be enzymes critical for the initiation and completion of DNA replication and cell division from yeast to mammals (1-5). The activity of CDKs is modulated through phosphorylation of their catalytic subunits and by association with activating or inhibiting proteins. For example, mitotic transition is mediated by the cdc2 kinase͞cyclin B complex, and activated cdc2 kinase has been found to drive dramatic structural reorganizations in the nuclear envelope, spindle apparatus, and chromosomal DNA by phosphorylation of a variety of substrates including histone, Eg5, lamin, vimentin, and plectin (6-12).Evidence suggests that serine͞threonine protein phosphatases function as crucial regulators of cell proliferation (4, 13-15). In particular, protein phosphatase 1 (PP-1), which is highly conserved in all eukaryotes, has been found to play a pivotal role in the cell cycle. Genetic studies have indicated that mutation of the enzyme in yeast, Aspergillus, and in Drosophila leads to a variety of defects in mitosis (16-18). Microinjection of anti-PP-1 antibody into B cells before cell division arrests cells at metaphase, whereas injection of PP-1 into anaphase cells accelerates cytokinesis (19). Expression of inhibitor-2, a specific PP-1 inhibitor, changes during the cell cycle, peaking during S phase and mitosis (20). Thus, the tightly balanced activity of CDKs and PP-1 appears to be required for normal cell division. However, the substrates that are targets f...

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Section: Discussionmentioning

confidence: 65%

Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase

Kwon

Lee

Choi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

204

192

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“…The resulting peptides were analyzed by mass spectrometry (16), which identified the 80 -90-kDa protein as the short form of ILF3 (also called NF90, NFAR-1, MPP4, and DRBP76). ILF3 is a nuclear protein that contains two dsRBD and an RGG domain and that influences gene expression (11,(17)(18)(19)(20)(21)(22). Importantly, the RNA/protein complex was dissociated upon addition of anti-ILF3 antibodies whereas mock antibodies had no effect (Fig.…”

Section: Ilf3mentioning

confidence: 99%

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

Gwizdek

Ossareh-Nazari

Brownawell

et al. 2004

Journal of Biological Chemistry

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The karyopherin-related nuclear transport factor exportin-5 preferentially recognizes and transports RNAs containing minihelix motif, a structural cis-acting export element that comprises a double-stranded stem (>14 nucleotides) with a base-paired 5 end and a 3-8-nucleotide protruding 3 end. This structural motif is present in various small cellular and viral polymerase III transcripts such as the adenovirus VA1 RNA (VA1). Here we show that the double-stranded RNA-binding protein, ILF3 (interleukin enhancer binding factor 3) preferentially binds minihelix motif. Gel retardation assays and glutathione S-transferase pull-down experiments revealed that ILF3, exportin-5, RanGTP, and VA1 RNA assembled in a quaternary complex in which the RNA moiety bridges the interaction between ILF3 and exportin-5. Formation of this complex is facilitated by the ability of both exportin-5 and ILF3 to mutually increase their apparent affinity for VA1 RNA. Using microinjection in the nucleus of HeLa cells and transfection experiments, we show here that formation of the cooperative RanGTP-dependent RNA/ILF3/exportin-5 complex promotes the co-transport of VA1 and ILF3 from the nucleus to the cytoplasm. Exportin-5 thus appears as the first example of a nuclear export receptor that mediates RNA export but also promotes transport of proteinaceous cargo through appropriate and specific RNA adaptors.Transfer of macromolecules through nuclear pore complexes depends on both structural components of nuclear pore complexes and soluble factors that ensure nuclear transport of a wide diversity of cellular proteins and RNAs. Selectivity of transport is mediated by the recognition of specific signals within the cargoes by specific transport receptors through a direct or adaptor-mediated interaction. Transport receptors interact with nucleoporins thereby mediating docking to and translocation through the nuclear pore complexes. Most of the transport receptors belong to the karyopherin ␤ family (also known as importin ␤ family) and use the GTPase Ran to control cargo association. The asymmetric distribution of the Ran regulatory proteins provides a steep gradient of RanGDP (cytoplasmic)/RanGTP (nuclear) across the nuclear envelope that ensures the directionality of nuclear transport (1, 2). Indeed, nuclear import receptors (importins) bind their cargoes in the cytoplasm in the absence of RanGTP and unload them upon binding to RanGTP in the nucleus. In contrast, export receptors (exportins) require RanGTP to interact with their cargoes in the nucleus, and those export complexes are destabilized by dissociation of RanGTP and GTP hydrolysis in the cytoplasm (3, 4).Study of nuclear export pathways of diverse viral RNAs has greatly contributed to the identification of cellular factors implicated in nuclear transport processes. In particular, we recently reported nuclear export mechanisms of the adenovirus VA1 RNA (5, 6). This RNA is abundantly transcribed by RNA polymerase III and efficiently exported to the cytoplasm in the late phase of the viral infect...

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“…First, Pin2-induced apoptotic cells would have some mitosis-speci®c markers and, second, Pin2-induced apoptosis would be increased if cells are arrested at mitosis by other approaches, but decreased if cells are not allowed to enter mitosis. To examine the ®rst prediction, we stained Pin2-transfected cells with the phospho-speci®c MPM-2 monoclonal antibody because MPM-2 speci®-cally recognizes a subset of mitosis-speci®c phosphoproteins and has been widely used as a maker for mitotic cells (Davis et al, 1983;Matsumoto-Taniura et al, 1996;Vandre et al, 1986;Westendorf et al, 1994;Ya e et al, 1997). As shown in Figure 4c, most Pin2-expressing cells were strongly stained with MPM-2 20 ± 24 h after transfection.…”

Section: Overexpression Of Pin2 Induces Mitotic Entry and Apoptosismentioning

confidence: 99%

Telomeric protein Pin2/TRF1 induces mitotic entry and apoptosis in cells with short telomeres and is down-regulated in human breast tumors

et al. 2001

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Telomeres are essential for cell survival and have been implicated in the mitotic control. The telomeric protein Pin2/TRF1 controls telomere elongation and its expression is tightly regulated during cell cycle. We previously reported that overexpression of Pin2/TRF1 a ects mitotic progression. However, the role of Pin2/TRF1 at the interface between cell division and cell survival remains to be determined. Here we show that overexpression of Pin2 induced apoptosis in cells containing short telomeres, but not in cells with long telomeres. Furthermore, before entering apoptosis, Pin2-expressing cells ®rst accumulated in mitosis and strongly stained with the mitosis-speci®c MPM2 antibody. Moreover, Pin2-induced apoptosis is potentiated by arresting cells in mitosis, but suppressed by accumulating cells in G1. In addition, overexpression of Pin2 also resulted in activation of caspase-3, and its proapoptotic activity was signi®cantly reduced by inhibition of caspase-3. These results indicate that up-regulation of Pin2/TRF1 can speci®cally induce entry into mitosis and apoptosis, likely via a mechanism related to activation of caspase-3. Signi®cantly, we also found that, out of 51 human breast cancer tissues and 10 normal controls examined, protein levels of Pin2/TRF1 in tumors were signi®cantly lower than in normal tissues, as detected by immunoblotting analysis and immunocytochemistry. Since down-regulation of Pin2/TRF1 allows cells to maintain long telomeres, these results suggest that down-regulation of Pin2/TRF1 may be important for cancer cells to extend their proliferative potential. Oncogene (2001) 20, 1497 ± 1508.

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Identification of novel M phase phosphoproteins by expression cloning.

Cited by 175 publications

References 44 publications

Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase

Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

Telomeric protein Pin2/TRF1 induces mitotic entry and apoptosis in cells with short telomeres and is down-regulated in human breast tumors

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