scholarly journals Phosphoinositide 3-Kinase Activation in Late G1 Is Required for c-Myc Stabilization and S Phase Entry

2006 ◽  
Vol 26 (23) ◽  
pp. 9116-9125 ◽  
Author(s):  
Amit Kumar ◽  
Miriam Marqués ◽  
Ana C. Carrera
Keyword(s):  
S Phase ◽  
Minichromosome Maintenance ◽  
Kinase Activation ◽  
Cell Cycle Entry ◽  
Pi3k Activity ◽  
Activity Peak ◽  
Pi3k Activation ◽  
Minichromosome Maintenance Protein ◽  
Phosphoinositide 3 Kinase ◽  
Phase Entry

ABSTRACT Phosphoinositide 3-kinase (PI3K) is one of the early-signaling molecules induced by growth factor (GF) receptor stimulation that are necessary for cell growth and cell cycle entry. PI3K activation occurs at two distinct time points during G1 phase. The first peak is observed immediately following GF addition and the second in late G1, before S phase entry. This second activity peak is essential for transition from G1 to S phase; nonetheless, the mechanism by which this peak is induced and regulates S phase entry is poorly understood. Here, we show that activation of Ras and Tyr kinases is required for late-G1 PI3K activation. Inhibition of late-G1 PI3K activity results in low c-Myc and cyclin A expression, impaired Cdk2 activity, and reduced loading of MCM2 (minichromosome maintenance protein) onto chromatin. The primary consequence of inhibiting late-G1 PI3K was c-Myc destabilization, as conditional activation of c-Myc in advanced G1 as well as expression of a stable c-Myc mutant rescued all of these defects, restoring S phase entry. These results show that Tyr kinases and Ras cooperate to induce the second PI3K activity peak in G1, which mediates initiation of DNA synthesis by inducing c-Myc stabilization.

scholarly journals Phosphoinositide 3-Kinases p110α and p110β Regulate Cell Cycle Entry, Exhibiting Distinct Activation Kinetics in G1 Phase

2008 ◽  
Vol 28 (8) ◽  
pp. 2803-2814 ◽  
Author(s):  
Miriam Marqués ◽  
Amit Kumar ◽  
Isabel Cortés ◽  
Ana Gonzalez-García ◽  
Carmen Hernández ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinases ◽  
Control Cell ◽  
Growth Factor Receptor ◽  
Maximum Activity ◽  
S Phase ◽  
Selective Action ◽  
Cell Cycle Entry ◽  
Phosphoinositide 3 Kinase ◽  
Phase Entry

ABSTRACT Phosphoinositide 3-kinase (PI3K) is an early signaling molecule that regulates cell growth and cell cycle entry. PI3K is activated immediately after growth factor receptor stimulation (at the G0/G1 transition) and again in late G1. The two ubiquitous PI3K isoforms (p110α and p110β) are essential during embryonic development and are thought to control cell division. Nonetheless, it is presently unknown at which point each is activated during the cell cycle and whether or not they both control S-phase entry. We found that p110α was activated first in G0/G1, followed by a minor p110β activity peak. In late G1, p110α activation preceded that of p110β, which showed the maximum activity at this time. p110β activation required Ras activity, whereas p110α was first activated by tyrosine kinases and then further induced by active Ras. Interference with p110α and -β activity diminished the activation of downstream effectors with different kinetics, with a selective action of p110α in blocking early G1 events. We show that inhibition of either p110α or p110β reduced cell cycle entry. These results reveal that PI3Kα and -β present distinct activation requirements and kinetics in G1 phase, with a selective action of PI3Kα at the G0/G1 phase transition. Nevertheless, PI3Kα and -β both regulate S-phase entry.

scholarly journals Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

2019 ◽  
Vol 218 (7) ◽  
pp. 2169-2184 ◽  
Author(s):  
Jacob Peter Matson ◽  
Amy M. House ◽  
Gavin D. Grant ◽  
Huaitong Wu ◽  
Joanna Perez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Live Cell Imaging ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycle Entry ◽  
Origin Licensing ◽  
Dna Replication Origins ◽  
Phase Entry

To maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is minichromosome maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading before S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. Consequently, the first S phase cells are hypersensitive to replication stress. This underlicensing results from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher-risk first cell cycle that likely promotes genome instability.

scholarly journals Phosphoinositide 3-kinase β regulates chromosome segregation in mitosis

2012 ◽  
Vol 23 (23) ◽  
pp. 4526-4542 ◽  
Author(s):  
Virginia Silió ◽  
Javier Redondo-Muñoz ◽  
Ana C. Carrera
Keyword(s):  
Chromosome Segregation ◽  
Genomic Stability ◽  
Pi3k Pathway ◽  
S Phase ◽  
Spindle Orientation ◽  
Aurora B ◽  
Pi3k Activity ◽  
Optimal Function ◽  
Phosphoinositide 3 Kinase ◽  
Phase Entry

Class IA phosphoinositide 3-kinases (PI3K) are enzymes composed of a p85 regulatory and a p110 catalytic subunit that control formation of 3-poly-phosphoinositides (PIP3). The PI3K pathway regulates cell survival, migration, and division, and is mutated in approximately half of human tumors. For this reason, it is important to define the function of the ubiquitous PI3K subunits, p110α and p110β. Whereas p110α is activated at G1-phase entry and promotes protein synthesis and gene expression, p110β activity peaks in S phase and regulates DNA synthesis. PI3K activity also increases at the onset of mitosis, but the isoform activated is unknown; we have examined p110α and p110β function in mitosis. p110α was activated at mitosis entry and regulated early mitotic events, such as PIP3 generation, prometaphase progression, and spindle orientation. In contrast, p110β was activated near metaphase and controlled dynein/dynactin and Aurora B activities in kinetochores, chromosome segregation, and optimal function of the spindle checkpoint. These results reveal a p110β function in preserving genomic stability during mitosis.

scholarly journals Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

2007 ◽  
Vol 18 (10) ◽  
pp. 4085-4095 ◽  
Author(s):  
Sharbani Chattopadhyay ◽  
Anja-Katrin Bielinsky
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
Catalytic Subunit ◽  
Drosophila Embryo ◽  
Minichromosome Maintenance ◽  
Α Subunit ◽  
Phase Entry

In Saccharomyces cerevisiae, minichromosome maintenance protein (Mcm) 10 interacts with DNA polymerase (pol)-α and functions as a nuclear chaperone for the catalytic subunit, which is rapidly degraded in the absence of Mcm10. We report here that the interaction between Mcm10 and pol-α is conserved in human cells. We used a small interfering RNA-based approach to deplete Mcm10 in HeLa cells, and we observed that the catalytic subunit of pol-α, p180, was degraded with similar kinetics as Mcm10, whereas the regulatory pol-α subunit, p68, remained unaffected. Simultaneous loss of Mcm10 and p180 inhibited S phase entry and led to an accumulation of already replicating cells in late S/G2 as a result of DNA damage, which triggered apoptosis in a subpopulation of cells. These phenotypes differed considerably from analogous studies in Drosophila embryo cells that did not exhibit a similar arrest. To further dissect the roles of Mcm10 and p180 in human cells, we depleted p180 alone and observed a significant delay in S phase entry and fork progression but little effect on cell viability. These results argue that cells can tolerate low levels of p180 as long as Mcm10 is present to “recycle” it. Thus, human Mcm10 regulates both replication initiation and elongation and maintains genome integrity.

scholarly journals Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.

1996 ◽  
Vol 16 (3) ◽  
pp. 1189-1202 ◽  
Author(s):  
A M Bennett ◽  
S F Hausdorff ◽  
A M O'Reilly ◽  
R M Freeman ◽  
B G Neel
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Map Kinase ◽  
S Phase ◽  
Immediate Early ◽  
Kinase Activation ◽  
Epidermal Growth ◽  
Tyrosyl Phosphorylation ◽  
Phase Entry

Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable.

scholarly journals Homeostatic control of START through negative feedback between Cln3-Cdk1 and Rim15/Greatwall kinase in budding yeast

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Nicolas Talarek ◽  
Elisabeth Gueydon ◽  
Etienne Schwob
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Cell Size ◽  
Negative Feedback ◽  
S Phase ◽  
Homeostatic Control ◽  
Cell Cycle Entry ◽  
Greatwall Kinase ◽  
E2f Transcription Factors ◽  
Phase Entry

How cells coordinate growth and division is key for size homeostasis. Phosphorylation by G1-CDK of Whi5/Rb inhibitors of SBF/E2F transcription factors triggers irreversible S-phase entry in yeast and metazoans, but why this occurs at a given cell size is not fully understood. We show that the yeast Rim15-Igo1,2 pathway, orthologous to Gwl-Arpp19/ENSA, is up-regulated in early G1 and helps promoting START by preventing PP2ACdc55 to dephosphorylate Whi5. RIM15 overexpression lowers cell size while IGO1,2 deletion delays START in cells with low CDK activity. Deletion of WHI5, CDC55 and ectopic CLN2 expression suppress the START delay of igo1,2∆ cells. Rim15 activity increases after cells switch from fermentation to respiration, where Igo1,2 contribute to chromosome maintenance. Interestingly Cln3-Cdk1 also inhibits Rim15 activity, which enables homeostatic control of Whi5 phosphorylation and cell cycle entry. We propose that Rim15/Gwl regulation of PP2A plays a hitherto unappreciated role in cell size homeostasis during metabolic rewiring of the cell cycle.

scholarly journals Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry

2021 ◽  
Author(s):  
Yoko Hayashi-Takanaka ◽  
Yuichiro Hayashi ◽  
Yasuhiro Hirano ◽  
Atsuko Miyawaki-Kuwakado ◽  
Yasuyuki Ohkawa ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
G1 Phase ◽  
Histone H4 ◽  
Replication Origins ◽  
Minichromosome Maintenance ◽  
Limiting Step ◽  
Mcm Helicase ◽  
Dna Replication Origins ◽  
Phase Entry

Replication of genomic DNA is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase on DNA replication origins during the G1 phase is essential in initiating DNA replication. Here, we show that stepwise loading of two hexamer complexes of MCM occurs during G1 progression in human cells. This transition from the single-to-double hexamer was associated with levels of methylation at lysine 20 of histone H4 (H4K20). A single hexamer of MCM complexes was loaded at the replication origins with the presence of H4K20 monomethylation (H4K20me1) in the early G1 phase, then another single hexamer was recruited to form a double hexamer later in G1 as H4K20me1 was converted to di-/tri-methylation (H4K20me2/me3). Under non-proliferating conditions, cells stay halted at the single-hexamer state in the presence of H4K20me1. We propose that the single-hexamer state on chromatin is a limiting step in making the proliferation-quiescence decision.

scholarly journals Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

2011 ◽  
Vol 192 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Marjorie A. Kuipers ◽  
Timothy J. Stasevich ◽  
Takayo Sasaki ◽  
Korey A. Wilson ◽  
Kristin L. Hazelwood ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Replication Fork ◽  
S Phase ◽  
G1 Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycles ◽  
Replication Arrest ◽  
Minichromosome Maintenance Protein ◽  
Mcm Proteins

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles.

scholarly journals Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

2019 ◽  
Author(s):  
Jacob Peter Matson ◽  
Amy M. House ◽  
Gavin D. Grant ◽  
Huaitong Wu ◽  
Joanna Perez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycle Entry ◽  
Origin Licensing ◽  
Dna Replication Origin ◽  
Dna Replication Origins

SUMMARYThe authors find that human cells re-entering the cell cycle from quiescence have both an impaired p53-dependent DNA replication origin licensing checkpoint and slow origin licensing. This combination makes every first S phase underlicensed and hypersensitive to replication stress.ABSTRACTTo maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is Minichromosome Maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading prior to S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. As a result, the first S phase cells are hypersensitive to replication stress. This underlicensing is from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher risk cell cycle that promotes genome instability.

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