CDC25A pathway toward tumorigenesis: Molecular targets of CDC25A in cell-cycle regulation

The cell division cycle 25 (CDC25) phosphatases regulate key transitions between cell-cycle phases during normal cell division, and in the case of DNA damage, they are key targets of the checkpoint machinery that ensure genetic stability. Little is known about the mechanisms underlying dysregulation and downstream targets of CDC25. To understand these mechanisms, we silenced the CDC25A gene in breast cancer cell line MDA-MB-231 and studied downstream targets of CDC25A gene. MDA-MB-231 breast cancer cells were transfected and silenced by CDC25A small interfering RNA. Total messenger RNA (mRNA) was extracted and analyzed by quantitative real-time polymerase chain reaction. CDC25A phosphatase level was visualized by Western blot analysis and was analyzed by 2D electrophoresis and LC-ESI-MS/MS. After CDC25A silencing, cell proliferation reduced, and the expression of 12 proteins changed. These proteins are involved in cell-cycle regulation, programmed cell death, cell differentiation, regulation of gene expression, mRNA editing, protein folding, and cell signaling pathways. Five of these proteins, including ribosomal protein lateral stalk subunit P0, growth factor receptor bound protein 2, pyruvate kinase muscle 2, eukaryotic translation elongation factor 2, and calpain small subunit 1 increase the activity of cyclin D1. Our results suggest that CDC25A controls the cell proliferation and tumorigenesis by a change in expression of proteins involved in cyclin D1 regulation and G1/S transition.     

Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response

BACKGROUND: Checkpoint signaling pathways are of crucial importance for the maintenance of genomic integrity. Within these pathways, the effector kinase Chk1 plays a central role in mediating cell-cycle arrest in response to DNA damage, and it does so by phosphorylating key cell-cycle regulators. RESULTS: By investigating the subcellular distribution of Chk1 by cell fractionation, we observed that around 20% of it localizes to chromatin during all phases of the cell cycle. Furthermore, we found that in response to DNA damage, Chk1 rapidly dissociates from the chromatin. Significantly, we observed a tight correlation between DNA-damage-induced Chk1 phosphorylation and chromatin dissociation, suggesting that phosphorylated Chk1 does not stably associate with chromatin. Consistent with these events being triggered by active checkpoint signaling, inhibition of the DNA-damage-activated kinases ATR and ATM, or siRNA-mediated downregulation of the DNA-damage mediator proteins Claspin and TopBP1, impaired DNA-damage-induced dissociation of Chk1 from chromatin. Finally, we established that Chk1 phosphorylation occurs at localized sites of DNA damage and that constitutive immobilization of Chk1 on chromatin results in a defective DNA-damage-induced checkpoint arrest. CONCLUSIONS: Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKK-dependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.     

Non-coding RNAs enter mitosis: functions, conservation and implications

Regulation of cytoskeletal remodeling is essential for cell cycle transitions. In fission yeast two NDR kinase signaling cascades, MOR and SIN, regulate the actin cytoskeleton to promote polarized growth during interphase and cytokinesis respectively. Our understanding of how these signaling pathways are coordinated to assist transition between the two cell-cycle stages is limited. Here, we review work from our laboratory, which reveals that cross talk between the SIN and MOR pathways is required for inhibition of interphase polarity programs during cytokinesis. Given the conservation of NDR kinase signaling pathways, our results may define general mechanisms by which these pathways are coordinated in higher organisms.     

Balancing of the mitotic exit network and cell wall integrity signaling governs the development and pathogenicity in Magnaporthe oryzae

The fungal cell wall plays an essential role in maintaining cell morphology, transmitting external signals, controlling cell growth, and even virulence. Relaxation and irreversible stretching of the cell wall are the prerequisites of cell division and development, but they also inevitably cause cell wall stress. Both Mitotic Exit Network (MEN) and Cell Wall Integrity (CWI) are signaling pathways that govern cell division and cell stress response, respectively, how these pathways cross talk to govern and coordinate cellular growth, development, and pathogenicity remains not fully understood. We have identified MoSep1, MoDbf2, and MoMob1 as the conserved components of MEN from the rice blast fungus Magnaporthe oryzae. We have found that blocking cell division results in abnormal CWI signaling. In addition, we discovered that MoSep1 targets MoMkk1, a conserved key MAP kinase of the CWI pathway, through protein phosphorylation that promotes CWI signaling. Moreover, we provided evidence demonstrating that MoSep1-dependent MoMkk1 phosphorylation is essential for balancing cell division with CWI that maintains the dynamic stability required for virulence of the blast fungus.     

When CDK1 rides the telomere cycle

Reconstitution of telomeric DNA at each cell division implies the coordination of DNA semiconservative replication with several processing events still poorly understood. Two reports published recently in Molecular Cell show that a cell-cycle cyclin-dependent kinase, Cdk1p, is required to create the cell-cycle-regulated overhang ().     

Mutations in N-cadherin and a Stardust homolog,Nagie oko,affect cell-cycle exit in zebrafish retina

It has been reported that the loss of apicobasal cell polarity and the disruption of adherens junctions induce hyperplasia in the mouse developing brain. However, it is not fully understood whether hyperplasia is caused by an enhanced cell proliferation, an inhibited neurogenesis, or both. In this study, we found that the ratio of the number of proliferating progenitor cells to the total number of retinal cells increases in the neurogenic stages in zebrafish n-cadherin (ncad) and nagie oko (nok) mutants, in which the apicobasal cell polarity and adherens junctions in the retinal epithelium are disrupted. The cell-cycle progression was not altered in the ncad and nok mutants. Rather, the ratio of the number of cells undergoing neurogenic cell division to the total number of cells undergoing mitosis decreased in the ncad and nok mutant retinas, suggesting that the switching from proliferative cell division to neurogenic cell division was compromised in these mutant retinas. These findings suggest that the inhibition of neurogenesis is a primary defect that causes hyperplasia in the ncad and nok mutant retinas. The Hedgehog-protein kinase A signaling pathway and the Notch signaling pathway regulate retinal neurogenesis in zebrafish. We found that both signaling pathways are involved in the generation of neurogenic defects in the ncad and nok mutant retinas. Taken together, these findings suggest that apicobasal cell polarity and epithelial integrity are essential for retinal neurogenesis in zebrafish.     

Extracellular control of cell size

Both cell growth (cell mass increase) and progression through the cell division cycle are required for sustained cell proliferation. Proliferating cells in culture tend to double in mass before each division, but it is not known how growth and division rates are co-ordinated to ensure that cell size is maintained. The prevailing view is that coordination is achieved because cell growth is rate-limiting for cell-cycle progression. Here, we challenge this view. We have investigated the relationship between cell growth and cell-cycle progression in purified rat Schwann cells, using two extracellular signal proteins that are known to influence these cells. We find that glial growth factor (GGF) can stimulate cell-cycle progression without promoting cell growth. We have used this restricted action of GGF to show that, for cultured Schwann cells, cell growth rate alone does not determine the rate of cell-cycle progression and that cell size at division is variable and depends on the concentrations of extracellular signal proteins that stimulate cell-cycle progression, cell growth, or both.     

Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.     

Oncogenic Met receptor induces cell-cycle progression in Xenopus oocytes independent of direct Grb2 and Shc binding or Mos synthesis, but requires phosphatidylinositol 3-kinase and Raf signaling

Biological responses of hepatocyte growth factor (HGF) are mediated by the Met receptor tyrosine kinase. Although HGF is a potent mitogen for a variety of cells, the signals required for cell-cycle progression by the Met/HGF receptor are poorly defined. In this study, we have used the Xenopus oocyte system to define the role of various Met proximal-binding partners and downstream signaling pathways in cell-cycle regulation. We show that cell-cycle progression and activation of MAPK and JNK mediated by the oncogenic Met receptor, Tpr-Met, are dependent on its kinase activity and the presence of the twin phosphotyrosine (Y482 & Y489) residues in its C-terminus, but that the recruitment of Grb2 and Shc adaptor proteins is dispensable, implicating other signaling molecules. However, using Met receptor oncoproteins engineered to recruit specific signaling proteins, we demonstrate that recruitment of Grb2 or Shc adaptor proteins is sufficient to induce cell-cycle progression and activation of MAPK and JNK, while the binding of phospholipase-Cgamma or phosphatidylinositol 3-kinase alone fails to elicit these responses. Using various means to block phosphatidylinositol 3-kinase, phospholipase-Cgamma, MEK, JNK, Mos, and Raf1 activity, we show that unlike the fibroblast growth factor receptor, MEK-dependent and independent signaling contribute to Met receptor-mediated cell-cycle progression, but phospholipase-Cgamma or JNK activity and Mos synthesis are not critical. Notably, we demonstrate that Raf1 and phosphatidylinositol 3-kinase signaling are required for cell-cycle progression initiated by the Met receptor, a protein frequently deregulated in human tumors.     

Plant casein kinases phosphorylate and destabilize a cyclin-dependent kinase inhibitor to promote cell division

Cell cycle is one of the most fundamentally conserved biological processes of plants and mammals. Casein kinase1s (CK1s) are critical for cell proliferation in mammalian cells; however, how CK1s coordinate cell division in plants remains unknown. Through genetic and biochemical studies, here we demonstrated that plant CK1, Arabidopsis (Arabidopsis thaliana) EL1-like (AELs), regulate cell cycle/division by modulating the stability and inhibitory effects of Kip-related protein6 (KRP6) through phosphorylation. Cytological analysis showed that AELs deficiency results in suppressed cell-cycle progression mainly due to the decreased DNA replication rate at S phase and increased period of G2 phase. AELs interact with and phosphorylate KRP6 at serines 75 and 109 to stimulate KRP6’s interaction with E3 ligases, thus facilitating the KRP6 degradation through the proteasome. These results demonstrate the crucial roles of CK1s/AELs in regulating cell division through modulating cell-cycle rates and elucidate how CK1s/AELs regulate cell division by destabilizing the stability of cyclin-dependent kinase inhibitor KRP6 through phosphorylation, providing insights into the plant cell-cycle regulation through CK1s-mediated posttranslational modification.

Plant casein kinases coordinate cell cycle by regulating the stability of a cyclin-dependent kinase inhibitor through promoting interaction with E3 ubiquitin ligases and proteasomal degradation by phosphorylation.     

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

The cyclin-dependent kinase inhibitors,cki-1 and cki-2, act in overlapping but distinct pathways to control cell-cycle quiescence during C. elegans development

Cyclin-dependent kinase inhibitors (CKIs) are major contributors to the decision to enter or exit the cell cycle. The Caenorhabditis elegans genome encodes two CKIs belonging to the Cip/Kip family, cki-1 and cki-2. cki-1 has been shown to act as a canonical negative regulator of cell-cycle entry, while the role of cki-2 remains unclear. We identified cki-2 in a genome-wide RNAi screen to reveal genes essential for developmental cell-cycle quiescence. Examination of cki-2 knockout animals revealed extra rounds of cell divisions, verifying a role in establishing or maintaining the temporary cell-cycle arrest. Despite the overlapping defects, the pathways mediated by cki-1 and cki-2 are discrete since the extra cell phenotype conferred by a putative cki-2(null) mutation is enhanced upon additional loss of cki-1 activity. Moreover, the extra cell division defect of cki-2 is not increased with the additional loss of lin-35 Rb, as is seen with cki-1. Thus, both cki-1 and cki-2 mediate cell-cycle quiescence, but our genetic and phenotypic analyses demonstrate that they act within distinct pathways to exert control over the cell-cycle machinery.     

At the crossroads of differentiation and proliferation: precise control of cell-cycle changes by multiple signaling pathways in Drosophila follicle cells

Here, we discuss the findings to date about genes and pathways required for regulation of somatic follicle-cell proliferation and differentiation during Drosophila oogenesis and demonstrate how loss of these genes contributes to the tumorigenic potential of mutant cells. Follicle cells undergo cell-fate determination through stepwise activation of multiple signaling pathways, including the Notch, Hedgehog, Wingless, janus kinase/STAT, and JNK pathways. In addition, changes in DNA replication and cellular growth depend on the spatial and temporal activation of the mitotic cycle-endocycle and endocycle-gene amplification cell-cycle switches and insulin-dependent monitoring of cellular health; systemic loss of these pathways contributes to loss of controlled cellular proliferation, loss of differentiation/growth, and aberrant cell polarity in follicle cells. We also highlight the effects of the neoplastic and Hippo pathways on the cell cycle and cellular proliferation in promoting normal development and conclude that lack of coordination of multiple signaling pathways promotes conditions favorable for tumorigenesis.     

RASSF1A suppresses melanoma development by modulating apoptosis and cell-cycle progression

The tumor suppressor candidate gene Ras association domain family 1, isoform A (RASSF1A) encodes a microtubule-associated protein that is implicated in the regulation of cell proliferation, migration, and apoptosis. Several studies indicate that down-regulation of RASSF1A resulting from promoter hypermethylation is a frequent epigenetic abnormality in malignant melanoma. In this study, we report that compared with melanocytes in normal skins or benign skin lesions, RASSF1A is down-regulated in melanoma tissues as well as cell lines, and its expression negatively correlates with lymph node metastasis. Following ectopic expression in RASSF1A-deficient melanoma A375 cell line, RASSF1A reduces cell viability, suppresses cell-cycle progression but enhances apoptotic cell death. In vivo, RASSF1A expression inhibits the tumorigenic potential of A375 cells in nude mice, which also correlates with decreased cell proliferation and increased apoptosis. On the molecular level, ectopic RASSF1A expression leads to differential expression of 209 genes, including 26 down-regulated and 183 up-regulated ones. Among different signaling pathways, activation of the apoptosis signal-regulating kinase 1 (ASK1)/p38 MAP kinase signaling is essential for RASSF1A-induced mitochondrial apoptosis, and the inhibition of the Akt/p70S6 kinase/eIF4E signaling is also important for RASSF1A-mediated apoptosis and cell-cycle arrest. This is the first study exploring the biological functions and the underlying mechanisms of RASSF1A during melanoma development. It also identifies potential targets for further diagnosis and clinical therapy.     

Raptor is Phosphorylated by cdc2 during Mitosis

Background

The appropriate control of mitotic entry and exit is reliant on a series of interlocking signaling events that coordinately drive the biological processes required for accurate cell division. Overlaid onto these signals that promote orchestrated cell division are checkpoints that ensure appropriate mitotic spindle formation, a lack of DNA damage, kinetochore attachment, and that each daughter cell has the appropriate complement of DNA. We recently discovered that AMP-activated protein kinase (AMPK) modulates the G2/M phase of cell cycle progression in part through its suppression of mammalian target of rapamycin (mTOR) signaling. AMPK directly phosphorylates the critical mTOR binding partner raptor inhibiting mTORC1 (mTOR-raptor rapamycin sensitive mTOR kinase complex 1). As mTOR has been previously tied to mitotic control, we examined further how raptor may contribute to this process.

Methodology/Principal Findings

We have discovered that raptor becomes highly phosphorylated in cells in mitosis. Utilizing tandem mass spectrometry, we identified a number of novel phosphorylation sites in raptor, and using phospho-specific antibodies demonstrated that raptor becomes phosphorylated on phospho-serine/threonine-proline sites in mitosis. A combination of site-directed mutagenesis in a tagged raptor cDNA and analysis with a series of new phospho-specific antibodies generated against different sites in raptor revealed that Serine 696 and Threonine 706 represent two key sites in raptor phosphorylated in mitosis. We demonstrate that the mitotic cyclin-dependent kinase cdc2/CDK1 is the kinase responsible for phosphorylating these sites, and its mitotic partner Cyclin B efficiently coimmunoprecipitates with raptor in mitotic cells.

Conclusions/Significance

This study demonstrates that the key mTOR binding partner raptor is directly phosphorylated during mitosis by cdc2. This reinforces previous studies suggesting that mTOR activity is highly regulated and important for mitotic progression, and points to a direct modulation of the mTORC1 complex during mitosis.     

Induction of postmitotic neuroretina cell proliferation by distinct Ras downstream signaling pathways

Ras-induced cell transformation is mediated through distinct downstream signaling pathways, including Raf, Ral-GEFs-, and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. In some cell types, strong activation of the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) cascade leads to cell cycle arrest rather than cell division. We previously reported that constitutive activation of this pathway induces sustained proliferation of primary cultures of postmitotic chicken neuroretina (NR) cells. We used this model system to investigate the respective contributions of Ras downstream signaling pathways in Ras-induced cell proliferation. Three RasV12 mutants (S35, G37, and C40) which differ by their ability to bind to Ras effectors (Raf, Ral-GEFs, and the p110 subunit of PI 3-kinase, respectively) were able to induce sustained NR cell proliferation, although none of these mutants was reported to transform NIH 3T3 cells. Furthermore, they all repressed the promoter of QR1, a neuroretina growth arrest-specific gene. Overexpression of B-Raf or activated versions of Ras effectors Rlf-CAAX and p110-CAAX also induced NR cell division. The mitogenic effect of the RasC40-PI 3-kinase pathway appears to involve Rac and RhoA GTPases but not the antiapoptotic Akt (protein kinase B) signaling. Division induced by RasG37-Rlf appears to be independent of Ral GTPase activation and presumably requires an unidentified mechanism. Activation of either Ras downstream pathway resulted in ERK activation, and coexpression of a dominant negative MEK mutant or mKsr-1 kinase domain strongly inhibited proliferation induced by the three Ras mutants or by their effectors. Similar effects were observed with dominant negative mutants of Rac and Rho. Thus, both the Raf-MEK-ERK and Rac-Rho pathways are absolutely required for Ras-induced NR cell division. Activation of these two pathways by the three distinct Ras downstream effectors possibly relies on an autocrine or paracrine loop, implicating endogenous Ras, since the mitogenic effect of each Ras effector mutant was inhibited by RasN17.