Human Progesterone Receptor Displays Cell Cycle-Dependent Changes in Transcriptional Activity

ABSTRACT The human progesterone receptor (PR) contains multiple Ser-Pro phosphorylation sites that are potential substrates for cyclin-dependent kinases, suggesting that PR activity might be regulated during the cell cycle. Using T47D breast cancer cells stably transfected with an mouse mammary tumor virus (MMTV) chloramphenicol acetyltransferase reporter (Cat0) synchronized in different phases of the cell cycle, we found that PR function and phosphorylation is remarkably cell cycle dependent, with the highest activity in S phase. Although PR expression was reduced in the G2/M phase, the activity per molecule of receptor was markedly reduced in both G1 and G2/M phases compared to the results seen with the S phase of the cell cycle. Although PR is recruited to the MMTV promoter equivalently in the G1 and S phases, recruitment of SRC-1, SRC-3, and, consequently, CBP is reduced in G1 phase despite comparable expression levels of SRC-1 and SRC-3. In G2/M phase, site-specific phosphorylation of PR at Ser162 and at Ser294, a site previously reported to be critical for transcriptional activity and receptor turnover, was abolished. Treatment with the histone deacetylase inhibitor trichostatin A elevated G1 and G2/M activity to that of the S phase, indicating that the failure to recruit sufficient levels of active histone acetyltransferase is the primary defect in PR-mediated transactivation.

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Review of: Human progesterone receptor displays cell cycle-dependent changes in transcriptional activity

Breast Cancer Online ◽

10.1017/s1470903106004834 ◽

2006 ◽

Vol 9 (02) ◽

Author(s):

C. A. Lange

Keyword(s):

Cell Cycle ◽

Progesterone Receptor ◽

Transcriptional Activity ◽

Human Progesterone Receptor ◽

Cycle Dependent

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Cell Cycle Dependent Regulation of Human Progesterone Receptor in Breast Cancer

10.21236/ada433995 ◽

2004 ◽

Author(s):

Lisa K. Mullany ◽

Carol A. Lange

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Progesterone Receptor ◽

Human Progesterone Receptor ◽

Cycle Dependent

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Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

Journal of Biological Chemistry ◽

10.1074/jbc.m111.338467 ◽

2012 ◽

Vol 287 (15) ◽

pp. 11891-11898 ◽

Cited By ~ 27

Author(s):

Kyung Yong Lee ◽

Sung Woong Bang ◽

Sang Wook Yoon ◽

Seung-Hoon Lee ◽

Jong-Bok Yoon ◽

...

Keyword(s):

Cell Cycle ◽

Origin Recognition Complex ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Replication Origins ◽

Eukaryotic Chromosome ◽

M Phase ◽

Cycle Dependent ◽

Prereplicative Complex ◽

Origin Recognition

During the late M to the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2–5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2–5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

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TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells

Journal of Cell Science ◽

10.1242/jcs.110.2.179 ◽

1997 ◽

Vol 110 (2) ◽

pp. 179-189 ◽

Cited By ~ 7

Author(s):

T. Asada ◽

R. Kuriyama ◽

H. Shibaoka

Keyword(s):

Cell Cycle ◽

Preprophase Band ◽

S Phase ◽

Motor Domain ◽

Bipolar Structure ◽

Amino Terminal ◽

M Phase ◽

Microtubule Motor ◽

Cycle Dependent ◽

In Cells

Analysis of a cDNA for a 125 kDa polypeptide, previously isolated from phragmoplasts of tobacco BY-2 cells as a candidate for a plus end-directed microtubule motor, revealed this polypeptide to be a novel member of the kinesin superfamily. We named this protein TKRP125 (tobacco kinesin-related polypeptide of 125 kDa). The strong similarity between TKRP125 and members of the bimC subfamily in terms of the amino acid sequence of the amino-terminal motor domain indicated that TKRP125 belonged to the bimC subfamily. An antibody against a short peptide from the motor domain of TKRP125 inhibited the GTP- or ATP-dependent translocation of phragmoplast microtubules in membrane-permeabilized BY-2 cells, suggesting a role for TKRP125 in microtubule translocation, which is considered to be involved in the formation and/or maintenance of the bipolar structure of the phragmoplast. The expression of TKRP125 was found to be cell cycle-dependent. TKRP125 was not present in cells at the G1 phase. It began to appear at the S phase and accumulated during the G2 phase. The distribution of TKRP125 changed as the arrangement of microtubules changed with the progression of the cell cycle. TKRP125 was distributed along cortical microtubules during the S phase and along microtubules in the preprophase band and perinuclear microtubules in premitotic cells. It was also present in the nucleus in premitotic cells. In cells in M phase, TKRP125 was distributed along spindle microtubules. It accumulated at the equatorial plane of the spindle as the spindle elongated. In cytokinetic cells, TKRP125 was colocalized with phragmoplast microtubules. These observations suggest the possible involvement of TKRP125 in the cell cycle-dependent changes in arrays of microtubules, including the organization of the phragmoplast, and in the movement of chromosomes in anaphase cells.

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Differential Localization and Activity of the A- and B-Forms of the Human Progesterone Receptor Using Green Fluorescent Protein Chimeras

Molecular Endocrinology ◽

10.1210/mend.13.3.0247 ◽

1999 ◽

Vol 13 (3) ◽

pp. 366-375 ◽

Cited By ~ 67

Author(s):

Carol S. Lim ◽

Christopher T. Baumann ◽

Han Htun ◽

Wenjuan Xian ◽

Masako Irie ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Progesterone Receptor ◽

Mammalian Cells ◽

Transcriptional Activity ◽

Fluorescent Protein ◽

Tumor Virus ◽

Fold Induction ◽

Differential Localization ◽

Human Progesterone Receptor ◽

Green Fluorescent

Abstract Subcellular localization and transcriptional activity of green fluorescent protein-progesterone receptor A and B chimeras (GFP-PRA and GFP-PRB) were examined in living mammalian cells. Both GFP-PRA and B chimeras were found to be similar in transcriptional activity compared with their non-GFP counterparts. GFP-PRA and PRA were both weakly active, while GFP-PRB and PRB gave a 20- to 40-fold induction using a reporter gene containing the full-length mouse mammary tumor virus long-terminal repeat linked to the luciferase gene (pLTRluc). Using fluorescence microscopy, nuclear/cytoplasmic distributions for the unliganded and hormone activated forms of GFP-PRA and GFP-PRB were characterized. The two forms of the receptor were found to have distinct intracellular distributions; GFP-PRA was found to be more nuclear than GFP-PRB in four cell lines examined. The causes for and implications of this differential localization of the A and B forms of the human PR are discussed.

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Cell Cycle-Dependent Expression of Mammalian E2-C Regulated by the Anaphase-Promoting Complex/Cyclosome

Molecular Biology of the Cell ◽

10.1091/mbc.11.8.2821 ◽

2000 ◽

Vol 11 (8) ◽

pp. 2821-2831 ◽

Cited By ~ 36

Author(s):

Atsushi Yamanaka ◽

Shigetsugu Hatakeyama ◽

Kin-ichiro Kominami ◽

Masatoshi Kitagawa ◽

Masaki Matsumoto ◽

...

Keyword(s):

Cell Cycle ◽

Substrate Specificity ◽

Critical Role ◽

Anaphase Promoting Complex ◽

Polyubiquitin Chain ◽

M Phase ◽

Ubiquitin Conjugating Enzyme ◽

Recognition Motifs ◽

Cycle Dependent ◽

Conjugating Enzyme

Progression through mitosis requires the precisely timed ubiquitin-dependent degradation of specific substrates. E2-C is a ubiquitin-conjugating enzyme that plays a critical role with anaphase-promoting complex/cyclosome (APC/C) in progression of and exit from M phase. Here we report that mammalian E2-C is expressed in late G2/M phase and is degraded as cells exit from M phase. The mammalian E2-C shows an autoubiquitinating activity leading to covalent conjugation to itself with several ubiquitins. The ubiquitination of E2-C is strongly enhanced by APC/C, resulting in the formation of a polyubiquitin chain. The polyubiquitination of mammalian E2-C occurs only when cells exit from M phase. Furthermore, mammalian E2-C contains two putative destruction boxes that are believed to act as recognition motifs for APC/C. The mutation of this motif reduced the polyubiquitination of mammalian E2-C, resulting in its stabilization. These results suggest that mammalian E2-C is itself a substrate of the APC/C-dependent proteolysis machinery, and that the periodic expression of mammalian E2-C may be a novel autoregulatory system for the control of the APC/C activity and its substrate specificity.

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Cell cycle–dependent localization of macroH2A in chromatin of the inactive X chromosome

The Journal of Cell Biology ◽

10.1083/jcb.200112074 ◽

2002 ◽

Vol 157 (7) ◽

pp. 1113-1123 ◽

Cited By ~ 74

Author(s):

Brian P. Chadwick ◽

Huntington F. Willard

Keyword(s):

Cell Cycle ◽

X Chromosome ◽

Degradation Pathway ◽

S Phase ◽

Histone H2a ◽

The Core ◽

Inactive X Chromosome ◽

Inactivation Center ◽

Chromatin Proteins ◽

Cycle Dependent

One of several features acquired by chromatin of the inactive X chromosome (Xi) is enrichment for the core histone H2A variant macroH2A within a distinct nuclear structure referred to as a macrochromatin body (MCB). In addition to localizing to the MCB, macroH2A accumulates at a perinuclear structure centered at the centrosome. To better understand the association of macroH2A1 with the centrosome and the formation of an MCB, we investigated the distribution of macroH2A1 throughout the somatic cell cycle. Unlike Xi-specific RNA, which associates with the Xi throughout interphase, the appearance of an MCB is predominantly a feature of S phase. Although the MCB dissipates during late S phase and G2 before reforming in late G1, macroH2A1 remains associated during mitosis with specific regions of the Xi, including at the X inactivation center. This association yields a distinct macroH2A banding pattern that overlaps with the site of histone H3 lysine-4 methylation centered at the DXZ4 locus in Xq24. The centrosomal pool of macroH2A1 accumulates in the presence of an inhibitor of the 20S proteasome. Therefore, targeting of macroH2A1 to the centrosome is likely part of a degradation pathway, a mechanism common to a variety of other chromatin proteins.

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Activity of the GR in G2 and Mitosis

Molecular Endocrinology ◽

10.1210/mend.16.6.0842 ◽

2002 ◽

Vol 16 (6) ◽

pp. 1352-1366 ◽

Cited By ~ 17

Author(s):

G. Alexander Abel ◽

Gabriela M. Wochnik ◽

Joëlle Rüegg ◽

Audrey Rouyer ◽

Florian Holsboer ◽

...

Keyword(s):

Cell Cycle ◽

Fluorescent Protein ◽

Chromatin Condensation ◽

Tyrosine Aminotransferase ◽

Cycle Phase ◽

M Cell ◽

Tumor Virus ◽

Mmtv Promoter ◽

Cycle Dependent ◽

Green Fluorescent

Abstract To elucidate the mechanisms mediating the reported transient physiological glucocorticoid resistance in G2/M cell cycle phase, we sought to establish a model system of glucocorticoid-resistant cells in G2. We synchronized various cell lines in G2 to measure dexamethasone (DEX)-induced transactivation of either two endogenous promoters (rat tyrosine aminotransferase and mouse metallothionein I) or the mouse mammary tumor virus (MMTV) promoter stably or transiently transfected. To circumvent the need for synchronization drugs, we stably transfected an MMTV-driven green fluorescent protein to directly correlate DEX-induced transactivation with the cell cycle position for each cell of an asynchronous population using flow cytometry. Surprisingly, all promoters tested were DEX-inducible in G2. Even in mitotic cells, only the stably transfected MMTV promoter was repressed, whereas the same promoter transiently transfected was inducible. The use of Hoechst 33342 for synchronization in previous studies probably caused a misinterpretation, because we detected interference of this drug with GR-dependent transcription independent of the cell cycle. Finally, GR activated a simple promoter in G2, excluding a functional effect of cell cycle-dependent phosphorylation of GR, as implied previously. We conclude that GR itself is fully functional throughout the entire cell cycle, but GR responsiveness is repressed in mitosis due to chromatin condensation rather than to specific modification of GR.

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