A novel ARF-binding protein (LZAP) alters ARF regulation of HDM2

The tumour suppressor ARF (alternative reading frame) is encoded by the INK4a (inhibitor of cyclin-dependent kinase 4)/ARF locus, which is frequently altered in human tumours. ARF binds MDM2 (murine double minute 2) and releases p53 from inhibition by MDM2, resulting in stabilization, accumulation and activation of p53. Recently, ARF has been found to associate with other proteins, but, to date, little is known about ARF-associated proteins that are implicated in post-translational regulation of ARF activity. Using a yeast two-hybrid screen, we have identified a novel protein, LZAP (LXXLL/leucine-zipper-containing ARF-binding protein), that interacts with endogenous ARF in mammalian cells. In the present study, we show that LZAP reversed the ability of ARF to inhibit HDM2's ubiquitin ligase activity towards p53, but simultaneously co-operated with ARF, maintaining p53 stability and increasing p53 transcriptional activity. Expression of LZAP, in addition to ARF, increased the percentage of cells in the G1 phase of the cell cycle. Expression of LZAP also caused activation of p53 and a p53-dependent G1 cell-cycle arrest in the absence of ARF. Taken together, our data suggest that LZAP can regulate ARF biochemical and biological activity. Additionally, LZAP has p53-dependent cell-cycle effects that are independent of ARF.

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Cooperative Signals Governing ARF-Mdm2 Interaction and Nucleolar Localization of the Complex

Molecular and Cellular Biology ◽

10.1128/mcb.20.7.2517-2528.2000 ◽

2000 ◽

Vol 20 (7) ◽

pp. 2517-2528 ◽

Cited By ~ 179

Author(s):

Jason D. Weber ◽

Mei-Ling Kuo ◽

Brian Bothner ◽

Enrico L. DiGiammarino ◽

Richard W. Kriwacki ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Ring Domain ◽

Negative Regulator ◽

Nucleolar Localization ◽

Subnuclear Localization ◽

Cycle Arrest ◽

Ubiquitin Ligase Activity ◽

Localization Signal ◽

Dependent Cell

ABSTRACT The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19 ARF and human p14 ARF bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19 ARF are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14 ARF protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19 ARF were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19 ARF are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19 ARF -Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.

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Methylation of histone H4 lysine 20 by PR-Set7 ensures the integrity of late replicating sequence domains in Drosophila

10.1101/028100 ◽

2015 ◽

Author(s):

Yulong Li () ◽

Robin L. Armstrong ◽

Robert J. Duronio ◽

David M. MacAlpine

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Cell Cycle Arrest ◽

Mammalian Cells ◽

Genome Stability ◽

Histone H4 ◽

Cycle Arrest ◽

Genomic Scale ◽

Dependent Cell

ABSTRACTThe methylation state of lysine 20 on histone H4 (H4K20) has been linked to chromatin compaction, transcription, DNA repair and DNA replication. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7. PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which has been partially attributed to defects in origin selection and activation. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 and H4K20 methylation impact the replication program on a genomic scale. We employed genetic, cytological, and genomic approaches to better understand the role of PR-Set7 and H4K20 methylation in regulating DNA replication and genome stability in Drosophila cells. We find that deregulation of H4K20 methylation had no impact on origin activation throughout the genome. Instead, depletion of PR-Set7 and loss of H4K20me1 results in the accumulation of DNA damage and an ATR-dependent cell cycle arrest. Coincident with the ATR-dependent cell cycle arrest, we find increased DNA damage that is specifically limited to late replicating regions of the Drosophila genome, suggesting that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains.

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SARP, a new alternatively spliced protein phosphatase 1 and DNA interacting protein

Biochemical Journal ◽

10.1042/bj20060600 ◽

2007 ◽

Vol 402 (1) ◽

pp. 187-196 ◽

Cited By ~ 18

Author(s):

Gareth J. Browne ◽

Margarida Fardilha ◽

Senga K. Oxenham ◽

Wenjuan Wu ◽

Nicholas R. Helps ◽

...

Keyword(s):

Protein Phosphatase ◽

Mammalian Cells ◽

Leucine Zipper ◽

Transforming Growth Factor ◽

Binding Protein ◽

Transforming Growth Factor Β ◽

Ankyrin Repeat ◽

Protein Phosphatase 1 ◽

Binding Motif ◽

Alternative Mrna Splicing

PP1 (protein phosphatase 1) is a ubiquitously expressed serine/threonine-specific protein phosphatase whose activity towards different substrates appears to be mediated via binding to specific proteins that play critical regulatory and targeting roles. In the present paper we report the cloning and characterization of a new protein, termed SARP (several ankyrin repeat protein), which is shown to interact with all isoforms of PP1 by a variety of techniques. A region encompassing a consensus PP1-binding motif in SARP (K354VHF357) modulates endogenous SARP–PP1 activity in mammalian cells. This SARP–PP1 interaction motif lies partially within the first ankyrin repeat in contrast with other proteins [53BP2 (p53 binding protein 2), MYPT1/M110/MBS (myosin binding protein of PP1) and TIMAP (transforming growth factor β inhibited, membrane-associated protein)], where a PP1-binding motif precedes the ankyrin repeats. Alternative mRNA splicing produces several isoforms of SARP from a single human gene at locus 11q14. SARP1 and/or SARP2 (92–95 kDa) are ubiquitously expressed in all tissues with high levels in testis and sperm, where they are shown to interact with both PP1γ1 and PP1γ2. SARP3 (65 kDa) is most abundant in brain where SARP isoforms interact with both PP1α and PP1γ1. SARP is highly abundant in the nucleus of mammalian cells, consistent with the putative nuclear localization signal at the N-terminus. The presence of a leucine zipper near the C-terminus of SARP1 and SARP2, and the binding of mammalian DNA to SARP2, suggests that SARP1 and SARP2 may be transcription factors or DNA-associated proteins that modulate gene expression.

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Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome

The Journal of Cell Biology ◽

10.1083/jcb.200104104 ◽

2001 ◽

Vol 154 (2) ◽

pp. 331-344 ◽

Cited By ~ 80

Author(s):

Daniel Kornitzer ◽

Rakefet Sharf ◽

Tamar Kleinberger

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Mammalian Cells ◽

Growth Arrest ◽

Anaphase Promoting Complex ◽

Reading Frame ◽

Cycle Growth ◽

M Phase ◽

Dependent Growth ◽

Synthetically Lethal

Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)–dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4–PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.

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Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5620 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5620-5631 ◽

Cited By ~ 159

Author(s):

B Shan ◽

X Zhu ◽

P L Chen ◽

T Durfee ◽

Y Yang ◽

...

Keyword(s):

Transcription Factor ◽

Mammalian Cells ◽

Leucine Zipper ◽

T Antigen ◽

Transactivation Domain ◽

Recognition Sequence ◽

Cellular Genes ◽

Genes Encoding ◽

Associated Proteins ◽

Transcription Factor E2f

The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Ap12, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Ap12 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Ap12 encodes a protein with properties known to be characteristic of transcription factor E2F.

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Interferon Regulatory Factor 1 (IRF-1) induces p21WAF1/CIP1 dependent cell cycle arrest and p21WAF1/CIP1 independent modulation of survivin in cancer cells

Cancer Letters ◽

10.1016/j.canlet.2011.12.027 ◽

2012 ◽

Vol 319 (1) ◽

pp. 56-65 ◽

Cited By ~ 20

Author(s):

Michaele J. Armstrong ◽

Michael T. Stang ◽

Ye Liu ◽

Jinbo Gao ◽

Baoguo Ren ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cancer Cells ◽

Interferon Regulatory Factor ◽

Regulatory Factor ◽

Cycle Arrest ◽

Interferon Regulatory Factor 1 ◽

Dependent Cell

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MdmX Protects p53 from Mdm2-Mediated Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.20.3.1001-1007.2000 ◽

2000 ◽

Vol 20 (3) ◽

pp. 1001-1007 ◽

Cited By ~ 142

Author(s):

Mark W. Jackson ◽

Steven J. Berberich

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Nuclear Export ◽

P53 Protein ◽

Ring Finger ◽

Ring Finger Domain ◽

Cycle Arrest ◽

Dependent Cell ◽

Potential Basis ◽

P53 Tumor Suppressor Protein

ABSTRACT The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.

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