Each week we showcase a hot new cell signaling article from a Nature Publishing Group journal. Free full text access to the paper will be maintained for three months, after which the paper will be accessible via the Research Library.
A positive feedback loop between reactive oxygen species (ROS) and protein kinase Cδ (PKCδ) cooperates with the retinoblastoma tumor suppressor pathway to irreversibly arrest senescent cells.
Keeping the cell cycle under control is essential for tissue homeostasis and the prevention of cancer development. Tumor suppressors such as retinoblastoma protein (pRb) and its activator p16INK4a induce cellular senescence — a terminal differentiation state in which cells are metabolically active but cease to divide indefinitely. In Nature Cell Biology, Eiji Hara and colleagues uncover a positive feedback loop between reactive oxygen species (ROS) and protein kinase Cδ (PKCδ) that irreversibly arrests the cell-cycle downstream of p16INK4a and pRb.
The authors activated both the pRb and p53 cell cycle arrest/senescence pathways by inactivating the temperature sensitive SV40 large T antigen. They found that high serum levels contributed to the induction of irreversible cell-cycle arrest, indicating that mitogenic signals cooperate with tumor suppressors in the induction of senescence.
Mitogenic signaling increases ROS levels, which are known to be involved in the onset of senescence. Interestingly, Hara and colleagues show that neutralizing the high ROS levels found in senescent cells with oxygen radical scavengers promoted a re-initiation of proliferation. As ROS activate PKCδ to further the production of oxygen radicals, a positive feedback loop that sustains high levels of ROS is established. In addition, they show that PKCδ blocks cell division, at least in part, by inducing proteasomal degradation of WARTS (also known as LATS), a mitotic exit network kinase required for cytokinesis.
ROS-PKCδ signaling was reduced upon prevention of cellular senescence by RNAi-mediated depletion of p16INK4a. Conversely, ROS-PKCδ signaling increased in senescent human diploid fibroblasts and upon RNAi-mediated depletion of DNA-binding protein-1 (DP1), an essential activator of the transcription factor E2F. Together, these findings indicate that the p16INK4a-pRb pathway causes cell-cycle arrest by blocking E2F activity, which not only prevents DNA replication but also activates ROS production by increasing the levels of manganese superoxide dismutase. This study sheds mechanistic insight into the effects of p16INK4a in aging, and more importantly, it paradoxically demonstrates a role for ROS as tumor suppressors, which may help to explain the conflicting results in the use of anti-oxidants in cancer prevention.
Monica Hoyos Flight Cell Migration Gateway
Original References:
Akiko Takahashi, Naoko Ohtani, Kimi Yamakoshi, Shin-ichi Iida, Hidetoshi Tahara, Keiko Nakayama, Keiichi I. Nakayama, Toshinori Ide, Hideyuki Saya & Eiji Hara Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence Nature Cell Biology8, 1291-1297 (2006) Full text | PDF | Subscribe to Nature Cell Biology
Matthew R. Ramsey & Norman E. Sharpless ROS as a tumour suppressor? Nature Cell Biology8, 1213-1215 (2006) Full text | PDF | Subscribe to Nature Cell Biology
Further reading:
Christian M Beausejour & Judith Campisi Balancing regeneration and cancer Nature443, 404-405 (28 Sep 2006) Full text | PDF | Subscribe to Nature
Neutrophil signaling: Integrin and Syk in an adaptor-coupled ITAM
The phosphorylation of adaptors containing the immunoreceptor tyrosine-based activation motif (ITAMs) by Src kinases is the first intracellular step of integrin signaling in neutrophils and macrophages.
The innate immune response — including the adhesion of phagocytes to invading pathogens and the release of antimicrobial proteins — is mediated by the binding of chemokines and growth factors to integrin transmembrane receptors. Syk tyrosine kinases are usually considered to be the first intracellular target of integrin signaling. However, Src family kinases phosphorylate ITAMs during the first signaling step of the adaptive immune response elicited by antigen binding to immune receptors such as the T-cell receptor (TCR), leading to the activation of Syk kinases. In Nature Immunology, Mócsai and colleagues demonstrate in vivo for the first time under physiological conditions that Src phosphorylation of the ITAM-containing adaptors DAP12 and FcRγ is also required for integrin signaling in neutrophils and macrophages.
Neutrophils from mice deficient in both DAP12 and FcRγ adaptor proteins (DF double-knockout) did not show integrin-mediated immune responses, including tumor necrosis factor α (TNF-α)-induced respiratory bursts. DF double-knockout neutrophils did not reveal defects in adhesion-independent responses to TNF-α, such as actin polymerization and p38 phosphorylation, showing that adhesion-dependent integrin signaling was specifically disrupted. Phosphorylated Syk could not be detected in DF double-knockout neutrophils nor in Src-kinase-null neutrophils, indicating a connection between adaptor function, Src activity and Syk phosphorylation.
To elucidate the molecular details of this connection, the authors performed rescue experiments with wild-type and mutant versions of Syk and DAP12 in Syk-knockout or DF-double-knockout neutrophils. In the mutants, DAP12 was not phosphorylated, and Syk did not bind to phosphorylated ITAMs. Neither of these mutants were able to restore a functional response in the DF double-knockout or Syk neutrophil knockouts. As mature neutrophils have a life span too limited for protein activity analysis, the authors engineered hematopoietic stem cells expressing a tag allowing their isolation after injection into lethally irradiated mice. Similar results were obtained with double knock-out macrophages, suggesting that ITAM-mediated integrin signaling may be a property of all phagocytic lymphocytes.
This study demonstrates that integrin signaling in neutrophils mirrors and macrophages immunoreceptor signaling, linking integrin clustering and phosphorylation of adaptor ITAMs by Src kinases to Syk kinase activation. In contrast to immunoreceptors, integrins are not directly associated with adaptor proteins, and future studies may reveal how activated integrin clusters signal to the adaptor proteins in this pathway.
Mirko von Elstermann Functional Glycomics Gateway
Original Article Mócsai A. et al Integrin signaling in neutrophils and macrophages uses adaptors containing immunoreceptor tyrosine-based activation motifs Nature Immunology7, 1326-1333 (2006) Full text | PDF | Subscribe to Nature Immunology
RTK/ERK signaling: Proximal effectors revealed
A functional RNAi screen designed to isolate proximal effectors of RTK signaling has uncovered novel regulators, many of which have functions assigned in other pathways.
Receptor tyrosine kinase (RTK) signaling through extracellular-signal-regulated kinases (ERKs) is crucial in many developmental and pathological settings. A large number of forward genetic screens and mammalian cell culture experiments have delineated the core components of this signaling cassette. However, components that act in a context specific manner can be overlooked in traditional forward genetic screens as they may lack a phenotype. As reported in this week's Nature, Adam Friedman and Norbert Perrimon have developed a unique phospho-specific antibody reporter of proximal pathway activity. The authors isolated over three hundred pathway regulators from a functional, genome-wide RNA interference (RNAi) screen in Drosophila.
Over twenty thousand predicted genes were targeted by RNAi in Drosophila S2 cells, 1,168 of which were regulated in response to insulin, which activates the ERK pathway. Whereas 56% of the genes isolated have no annotated molecular function, 23% have a homologue implicated in mammalian disease and many have roles in processes such as cytoskeletal maintenance, neurogenesis and cell proliferation. In general, trafficking negatively regulated RTK activation, whereas proteosome and ribosome components were positive regulators.
RTK/ERK regulators may act in a cell-type specific manner or in response to stimulation from a specific RTK; therefore a second round of screening was undertaken. 362 genes were targeted by RNAi in different cell lines exposed to various stimuli. 331 genes affected RTK signaling output in all settings studied. Many effectors of the cell-cycle, the transcriptional machinery as well as components of other signaling pathways were isolated. Notably D-jun and D-fos, well known targets of JNK and ERK MAPK signaling, inhibit proximal pathway activation.
To determine which regulators act proximally in the pathway, the authors screened validated interactors in S2 cells that conditionally express Rasv12. 85 genes suppressed ERK activation. Genes that reduce ERK activation by insulin, but not by Ras, may function proximally to Ras.
The authors focused on two previously uncharacterized genes; the positive acting ste20-like kinase dGCKIII, and the inhibitor dPPM1, a member of the PPM Ser/Thr phosphatase family. dGCKIII RNAi knockdown in the imaginal wing disc resulted in a loss of tissue due to reduced cell survival. Furthermore, Raf1 and protein phosphatase 2 co-immunoprecipitate with dGCKIII. Expression of dPPM1 reduced wing size and suppressed the Rasv12-induced rough eye phenotype. In addition, dPPM1 co-immunoprecipitated with ERK. Equivalent experiments in human cell lines indicate evolutionary conservation of these signaling components.
This study isolates novel, proximal components of the RTK/ERK signaling pathway and reveals many as having roles in other pathways.
Clare Garvey Signaling Gateway
Original Article Adam Friedman and Norbert Perrimon. A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling Nature444, 230-234 (9 November 2006) Full text | PDF | Subscribe to Nature
Retinoblastoma: Adding p53 to the mix
Retinoblastomas amplify inhibitors of the p53 signaling pathway leading to cell survival and tumor progression.
p53 and Retinoblastoma (Rb) were the first two tumor-suppressor genes discovered and both have subsequently been implicated in almost all cancers. Rb controls cell proliferation and p53 mediates cell cycle arrest or apoptosis in response to cellular stress. While p53 is inactivated almost universally in cancers, retinoblastomas — which are initiated by an Rb mutation — express wild-type p53. It was hypothesized that retinoblastoma arises from cells that are intrinsically resistant to death and are therefore able to bypass the p53 pathway. However, findings presented in Nature reveal that Rb-deficient retinoblastomas can undergo p53-mediated apoptosis and that the p53 pathway is inactivated through amplification of the p53 antagonists MDMX or MDM2.
p14ARF is a key player in the p53 cellular response pathway; upon cellular stress E2F activates p14ARF transcription leading to the inactivation of the ubiquitin ligase MDM2 and hence de-repression of p53-mediated apoptosis. The authors demonstrate that p14ARF expression is increased in retinoblastomas as well as in primary human fetal retinas after siRNA or conditional knockout of Rb. The MDM2-related gene MDMX was amplified in 65% of retinoblastoma samples analyzed, while the p53 pathway downstream of MDMX was intact in retinoblastoma cells.
While deletion of Rb and p107 leads to an increased susceptibility to retinoblastoma, additional deletion of p53 causes complete cancer penetrance with highly aggressive tumors. Transient MDMX overexpression in Rb;p107-deficient retinas increased proliferation and survival of the developing retinal cells that expressed the retinoblastoma marker Pax6. Rb siRNA knockdown in human embryonic retinas resulted in extensive apoptosis, which was alleviated by co-expression of MDMX. The use of mutant MDMX that is unable to bind p53 confirmed that the suppression of cell death was specific to the p53 pathway.
Nutlin-3, a small molecule inhibitor of the MDM protein-p53 interaction induces the p53 pathway and kills retinoblastoma cells with MDMX amplification in culture.
The long standing view that retinoblastoma cells are death-resistant and retain a functional p53 pathway has been challenged by these findings. Furthermore, nutlin-3 may provide an enticing alternative to chemotherapy for children with retinoblastoma, as well as other cancers with MDMX amplifications.
Clare Garvey Signaling Gateway
Original Article Nikia A. Laurie, et al. Inactivation of the p53 pathway in retinoblastoma p61 Nature444, 61-66 (2 November 2006) Full text | PDF | Subscribe to Nature
News and Views Valerie A. Wallace Cancer biology: Second step to retinal tumours Nature444, 45-46 (2 November 2006) Full text | PDF | Subscribe to Nature
