Differential regulation of cell migration and cell cycle progression by FAK complexes with Src, PI3K, Grb7 and Grb2 in focal contacts.

Focal adhesion kinase (FAK) is a key mediator of integrin signaling, which has been implicated in the regulation of cell migration and cell cycle progression. Using chimeric molecules that fuse the focal adhesion targeting (FAT) sequence directly to several signaling molecules, we investigated the potential role of FAK recruitments of signaling molecules to focal contacts in the regulation of cell migration and cell cycle progression. We found that fusion of FAT to Src, the p85 subunit of phosphatidylinositol 3-kinase, Grb7 and Grb2 resulted in the efficient focal adhesion targeting of these signaling molecules. We showed that expression of Src-FAT, p85-FAT, or Grb7-FAT, but not Grb2-FAT, each stimulated cell migration. Interestingly, tyrosine phosphorylation of paxillin, but not p130cas, was induced by expression of Src-FAT, suggesting a potential role of paxillin in mediating stimulation of cell migration by the chimeric molecule. In contrast, targeting of Grb2, but not Src, p85, or Grb7, to focal contacts increased cell cycle progression. Biochemical analyses correlated Erk activation by Grb2-FAT with its stimulation of cell cycle progression. Together, these results suggest that at least part of the role of FAK interaction with these signaling molecules is to recruit them to focal contacts and that distinct FAK signaling complexes are involved in the regulation of cell migration vs. cell cycle progression.     

Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1.

Activation of growth factor receptors by ligand binding initiates a cascade of events leading to cell growth and division. Progression through the cell cycle is controlled by cyclin-dependent protein kinases (Cdks), but the mechanisms that link growth factor signaling to the cell cycle machinery have not been established. We report here that Ras proteins play a key role in integrating mitogenic signals with cell cycle progression through G1. Ras is required for cell cycle progression and activation of both Cdk2 and Cdk4 until approximately 2 h before the G1/S transition, corresponding to the restriction point. Analysis of Cdk-cyclin complexes indicates that Ras signaling is required both for induction of cyclin D1 and for downregulation of the Cdk inhibitor p27KIP1. Constitutive expression of cyclin D1 circumvents the requirement for Ras signaling in cell proliferation, indicating that regulation of cyclin D1 is a critical target of the Ras signaling cascade.     

P27 expression is regulated by separate signaling pathways, downstream of Ras, in each cell cycle phase

The cyclin inhibitory protein p27Kip1 (p27) plays a vital role in regulating cell proliferation in response to the extracellular growth environment. Active proliferation requires the suppression of p27 levels throughout the cell cycle. Late in the cell cycle, p27 degradation requires phosphorylation of Thr 187 by cyclin dependent kinase 2, leading to recognition by the SCF ubiquitin ligase containing the Skp2 F-box protein. Suppression of p27 is also essential for cell proliferation early in the cell cycle, but this occurs independently of Skp2, whose expression is suppressed during G1 phase. In this study, we use a time lapse and quantitative imaging approach to study the connection between proliferative signaling and the degradation of p27 during each cell cycle period in actively cycling cells. Ras activity was required for the suppression of p27 levels throughout the cell cycle, but separate pathways downstream of Ras signaling were required in different cell cycle periods. For example, inhibitors of MEK and phosphatidylinositol-3-kinase induced p27 expression primarily in G1 phase, while inhibitors of AKT activity stimulated these levels primarily in S phase. Skp2 was expressed in a Ras-dependent manner at higher levels late in the cell cycle. Its ablation resulted in higher p27 levels primarily in G2 phase as expected. The fact that separate signaling pathways downstream of Ras function in each cell cycle phase to suppress p27 levels helps explain the vital connection between proliferative signaling, cell cycle control, and p27 expression.     

A quantitative model of the effect of unreplicated DNA on cell cycle progression in frog egg extracts

A critical goal in cell biology is to develop a systems-level perspective of eukaryotic cell cycle controls. Among these controls, a complex signaling network (called ‘checkpoints’) arrests progression through the cell cycle when there is a threat to genomic integrity such as unreplicated or damaged DNA. Understanding the regulatory principles of cell cycle checkpoints is important because loss of checkpoint regulation may be a requisite step on the roadway to cancer. Mathematical modeling has proved to be a useful guide to cell cycle regulation by revealing the importance of bistability, hysteresis and time lags in governing cell cycle transitions and checkpoint mechanisms. In this report, we propose a mathematical model of the frog egg cell cycle including effects of unreplicated DNA on progression into mitosis. By a stepwise approach utilizing parameter estimation tools, we build a model that is grounded in fundamental behaviors of the cell cycle engine (hysteresis and time lags), includes new elements in the signaling network (Myt1 and Chk1 kinases), and fits a large and diverse body of data from the experimental literature. The model provides a validated framework upon which to build additional aspects of the cell cycle checkpoint signaling network, including those control signals in the mammalian cell cycle that are commonly mutated in cancer.     

Fibronectin-mediated adhesion rescues cell cycle arrest induced by fibroblast growth factor-1 by decreased expression of P21cip/waf in human chondrocytes

Summary In chondrocytes, fibroblast growth factors (FGFs) inhibit chondrocytes proliferation by upregulation of the cell cycle inhibitor p21^cip/waf. In this report, we first investigated the roles of fibronectin (FN)-mediated cell adhesion in the modulation of FGF-1's antiproliferative function in chondrocytes. In this study, we found that FN-mediated signaling could rescue cell cycle arrest induced by FGF-1 in primary human chondrocytes. This prevention of cell cycle arrest induced by FGF-1 was due to the suppression of the cell cycle inhibitor p21^cip/waf expression on adhesion to FN and its downstream activation of signaling pathways. Finally, we showed that this rescue induced by FN-mediated adhesion is dependent on the extracellular regulated kinase (ERK) signaling pathway. Taken together, these studies support that, despite FGF-FGF receptor's growth-inhibitory function, the FN-mediated signaling can collaborate to compensate for its negative effect on chondrocytes proliferation, providing evidence for cross talk between signals emerging from these cell surface molecules in chondrocyte.     

Growth factor-dependent signaling and cell cycle progression

There are three central ideas contained within this review. Firstly, growth factor-stimulated signaling is not restricted to a 30–60 min window, but occurs at a much later time as well. Secondly, the second wave of signaling overlaps temporally with the cell cycle program and may be directly responsible for engaging it. Thirdly, the G1 to S interval appears to encompass two distinct phases of the cell cycle, during which the coordinated activation of distinct sets of signaling enzymes drives cell cycle progression. Each of these concepts is likely to initiate new investigation and hence provide additional insight into the fundamental question of how growth factors drive cell proliferation.     

Proliferating CD4+ T cells undergo immediate growth arrest upon cessation of TCR signaling in vivo

To investigate the role of TCR signaling in the exit of CD4+ T cells from cell cycle, we took advantage of a low frequency TEa T cell adoptive transfer technique as well as the Y-Ae mAb to interrupt Ag/MHC recognition before the completion of clonal expansion. Termination of TCR signaling after 36 h of Ag exposure caused an immediate reduction in cell size and deceleration of G1->SG2M phase cell cycle progression. As a consequence, clonal expansion in the absence of durable TCR signaling decreased by two-thirds. Thus, CD4+ T cells scan for the presence Ag throughout their clonal expansion response, and continuously adjust their rate of cell growth and G1->S phase transition to match their intensity of TCR signaling.     

Interplay between MEK-ERK signaling, cyclin D1, and cyclin-dependent kinase 5 regulates cell cycle reentry and apoptosis of neurons

In response to neurotoxic signals, postmitotic neurons make attempts to reenter the cell cycle, which results in their death. Although several cell cycle proteins have been implicated in cell cycle-related neuronal apoptosis (CRNA), the molecular mechanisms that underlie this important event are poorly understood. Here, we demonstrate that neurotoxic agents such as β-amyloid peptide cause aberrant activation of mitogen-activated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling, which promotes the entry of neurons into the cell cycle, resulting in their apoptosis. The MEK-ERK pathway regulates CRNA by elevating the levels of cyclin D1. The increase in cyclin D1 attenuates the activation of cyclin-dependent kinase 5 (cdk5) by its neuronal activator p35. The inhibition of p35-cdk5 activity results in enhanced MEK-ERK signaling, leading to CRNA. These studies highlight how neurotoxic signals reprogram and alter the neuronal signaling machinery to promote their entry into the cell cycle, which eventually leads to neuronal cell death.     

Cell cycle-related signaling pathways modulated by peripheral benzodiazepine receptor ligands in colorectal cancer cells

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce both apoptosis and G1/G0 cell cycle arrest in colorectal cancers. The signaling pathways leading to cell cycle arrest are still unknown. Using cDNA array technology, we identified signaling molecules involved in cell cycle arrest induced by the PBR ligands FGIN-1-27 and PK 11195. Differential gene expression was confirmed by semi-quantitative RT-PCR or Western blot analysis of gene products. The PBR ligand-mediated signaling involved the upregulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, cdc16, and the cell cycle inhibitors gadd45 and gadd153, the downregulation of the cyclins D1 and B1, as well as the inactivation of ERK1/2. The p21-deficient colorectal cancer cell line HCT116 p21-/- was significantly less sensitive to PBR ligands than the parental HCT116 wild-type cells, demonstrating the functional involvement of p21WAF1/CIP1 in PBR ligand-mediated G1 arrest. This study thus revealed PBR ligand-triggered signaling pathways leading to cell cycle arrest. Moreover, we showed the functional implication and interaction of differentially expressed gene products and provided a model of signaling pathways involved in PBR ligand-induced G1 arrest. These results form the basis for future PBR ligand-mediated therapeutic approaches.     

Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway

The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed.     

hnRNP A1 promotes keratinocyte cell survival post UVB radiation through PI3K/Akt/mTOR pathway

hnRNP A1 acts as a critical splicing factor in regulating many alternative splicing events in various physiological and pathophysiological progressions. hnRNP A1 is capable of regulating UVB-induced hdm2 gene alternative splicing according to our previous study. However, the biological function and underlying molecular mechanism of hnRNP A1 in cell survival and cell cycle in response to UVB irradiation are still unclear. In this study, silencing hnRNP A1 expression by siRNA transfection led to decreased cell survival after UVB treatment, while promoting hnRNP A1 by lentiviruse vector resulted in increased cell survival. hnRNP A1 remarkably enhanced PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of Akt, mTOR and P70S6 protein. Inhibition of PI3K/Akt signaling by LY294002 suppressed the expression of hnRNP A1. While mTOR signaling inhibitors, rapamycin and AZD8055, did not influence hnRNP A1 expression in HaCaT cells, suggesting that hnRNP A1 may be an upstream mediator of mTOR signaling. Furthermore, hnRNP A1 could alleviate UVB-provoked cell cycle arrest at G0/G1 phase and promoted cell cycle progression at G2/M phase. Our results indicate that hnRNP A1 promotes cell survival and cell cycle progression following UVB radiation.     

γH2AX as a Checkpoint Maintenance Signal

A key function of the conserved signaling cascade triggered by DNA damage is to promote the arrest or pause of cell cycle progression, a phenomenon commonly termed a “checkpoint”. This response allows time for DNA repair to proceed before entering a new phase of the cell cycle. Although much is known about the initiation and propagation of this signaling pathway, much less is understood about the mechanisms that lead to its extinction. Recent work highlights a role for H2AX phosphorylation as a checkpoint maintenance factor and of its dephosphorylation as a signal for resumption of the cell cycle.     

Cytosolic p21Waf1/Cip1 increases cell cycle transit in vascular smooth muscle cells

The intracellular localization of signaling proteins is critical in directing their interactions with both upstream and downstream signaling cascade components. While initially described as a cyclin kinase inhibitor, p21Waf1/Cip1 has since been shown to have bimodal effects on cell cycle progression and cell proliferation, and evidence is emerging that intracellular localization of this protein plays a role in directing its signaling properties by dictating its interactions with downstream molecules. Since we have previously demonstrated a pro-apoptotic and cell cycle inhibitory effect of p21 attenuation after transfection of antisense p21 oligodeoxynucleotides (ODN) in several cell lines, we asked whether cytosolic p21 mediates a positive effect on vascular smooth muscle (VSM) cell cycle transit. We now show that transfection of a nuclear-localization signal deficient (DeltaNLS) p21 construct into VSM cells results in increased cytosolic levels of p21 and causes increased cell cycle transit as measured by [3H]thymidine incorporation. Thus, at least in VSM cells, cytosolic localization of p21 is a means by which this signaling protein transmits pro-mitogenic signals to the proteins responsible for G1/S transition. Furthermore, compartmentalization of p21 may help explain the biphasic nature of p21 in a variety of cell types and may lead to therapeutic advances directed at modulating pathologic cell growth in vascular diseases and cancer.