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.
Loss of Ras binding to phosphoinositide 3-kinase γ (PI3Kγ) by mutating its catalytic subunit p110γ leads to diminished Drosophila cell growth and reduced migration of mouse neutrophils.
PI3Ks catalyze the formation of the second messenger lipid phosphatidylinositol (3,4,5) triphosphate (PIP3), consequently they are are among the most important drivers of cell proliferation and migration. They consist of a regulatory p85 subunit and a catalytic p110 subunit; the small GTPase Ras can bind and activate all PI3K subunits in vitro but the in vivo relevance of this interaction has been unclear. The PI3K subclass PI3Kγ also possesses a distinct regulatory subunit, p101, which participates in PI3Kγ activation by the heterotrimeric G-protein βγ-complex (Gβγ). In Nature Cell Biology, Orme et al. and Suire et al. now show that PI3Kγ requires Ras binding to p110γ for maximum activity.
To investigate the role of Ras in PI3Kγ signaling, both studies used flies and mice expressing mutant forms of p110 subunits that were unable to bind to Ras. Orme et al. looked at the effect of expressing Myc-Dp110&gammaRBD in flies. Mutant flies had a normal phenotype except for wing size and egg production, which was thought to depend mainly on activation of PI3K through insulin signaling. This study indicates that in Drosophila PI3Kγ activity requires Ras binding in addition to insulin signaling to achieve maximum cell growth and proliferation.
To compare the effect of both Ras and heterotrimeric G-protein binding in the regulation of PI3Kγ activity in mice, Suire et al. used animals expressing p110DASA/DASA — which can't bind Ras — and animals lacking the p101 regulatory unit (p101-/-). They found that G-protein activators, known for their effect on PI3Kγ activation, reduced the levels of PIP3 production significantly in both p110DASA/DASA as well as in p101-/- neutrophils. Neutrophil migration towards inflammation sites also depends on their capacity to respond to these G-protein activators and the percentage of p110DASA/DASA neutrophils that were able to move in a chemoattractant gradient was reduced up to 75%. These results reveal an unexpected synergistic effect of Ras and Gβγ binding on PI3Kγ activation. Surprisingly, p110DASA/DASA — but not p101-/- neutrophils — were found to have dramatically reduced production of Reactive Oxygen Species (ROS). As ROS is involved in the killing of pathogens by neutrophils this suggests a separation of the downstream effects of PI3K signaling, Ras or Gβγ binding.
The combined results of these two studies show how Ras can modulate PI3K activity in response to specific signaling cascades and in different cellular contexts.
Mirko von Elstermann Functional Glycomics Gateway
letter Sabine Suire, Alison M. Condliffe, G. John Ferguson, Chris D. Ellson, Hervé Guillou, Keith Davidson, Heidi Welch, John Coadwell, Martin Turner, Edwin R. Chilvers, Phillip T. Hawkins & Len Stephens> Gβγs and the Ras binding domain of p110γ are both important regulators of PI3Kγ signalling in neutrophils Nature Cell Biology(15 October 2006) Full text | PDF | Subscribe to Nature
letter Mariam H. Orme, Saif Alrubaie, Gemma L. Bradley, Cherryl D. Walker & Sally J. Leevers
Input from Ras is required for maximal PI(3)K signalling in Drosophila Nature (15 October 2006) Full text | PDF | Subscribe to Nature
Seed development: The hunt for the missing receptor ABAtes
The Mg-chelatase H subunit (CHLH) is the elusive abscisic acid receptor (ABAR) required for seed development and stomatal aperture regulation.
Absciscic acid (ABA) is a key phytohormone required for seed development and other developmental processes in plants. A study published in Nature last year revealed the RNA-binding protein FCA as the abscisic acid receptor required for regulating flowering time, however the receptor required for seed development and stomatal aperture regulation remained elusive. Now, Shen et al. have gone further by revealing the Mg-chelatase H subunit as the missing Abscisic acid receptor (ABAR) in this setting.
The authors previously described an ABA binding protein from broad bean that was implicated in stomatal signaling. They showed this protein to be the putative H subunit (CHLH) of the magnesium protoporphyrin-IX (Proto) chelatase (Mg-chelatase). In the latest study, the authors demonstrate that purified ABAR from Arabidopsis binds ABA in a highly stereospecific manner. Co-immunoprecipitation assays revealed binding activity proportional to the starting amount of ABAR protein. In order to assess the role that this receptor plays in plant development the authors make use of antisense and overexpression lines. RNAi knockdown of ABAR results in ABA-insensitive phenotypes in seed germination and inhibition of stomatal opening, while plants overexpressing ABAR displayed ABA-hypersensitive phenotypes such as increased resistance to leaf dehydration. The authors further describe a transferred DNA (T-DNA) insertion mutant in the ABAR gene that gives a seed phenotype similar to other ABA-signaling component seed maturation phenotypes.
Mg-chelatase is composed of three subunits and catalyzes a reaction crucial to chlorophyll synthesis. CHLH is a Proto-binding protein and has a previously described role in plastid-nucleus retrograde signaling. Treatment with ABA resulted in stimulated ABAR expression and Mg-chelatase activity. ABAR RNAi knockdown reduced the mRNA level of genes that respond positively to ABA. Analysis of mutants defective in chlorophyll metabolism or plastid signaling show that ABAR is a positive regulator of ABA signaling in a pathway that is distinct from that required for chlorophyll metabolism and Mg-Proto-mediated plastid signaling.
The authors show that CHLH is expressed not only in green but also in non-green tissue, including roots, implicating functionality for ABAR at the whole plant level. Indeed ABAR RNAi downregulation reduced levels of ABA responsive regulators in leaves and seeds. While ABAR is shown to be a receptor for ABA signaling, other components of this transduction pathway remain to be determined. ABAR is evolutionarily conserved and hints at putative roles in other organisms.
Clare Garvey Signaling Gateway
article Yuan-Yue Shen et al. The Mg-chelatase H subunit is an abscisic acid receptor Nature443, 823 - 826 (19 October 2006) Full text | PDF | Subscribe to Nature
Asymmetric targeting of β-actin mRNA in growth cones underlies axon turning in response to guidance cues.
The establishment of neuronal circuits requires the formation of appropriate connections between neurons. Axon guidance cues ensure that axons reach their targets but the changes within the growth cone that facilitate this process are poorly understood. Previous studies have shown that local actin synthesis and polymerization drives growth cone motility. Two studies in Nature Neuroscience now show that growth cone turning in response to brain-derived neurotrophic factor (BDNF) and netrin-1 requires asymmetric targeting of β-actin mRNA and local translation on one side of the growth cone.
James Zheng and colleagues show that inhibition of protein synthesis blocks BDNF-induced growth cone turning. What's more, local application of BDNF leads to an increase in the levels of β-actin mRNA on the side of the growth cone closest to the BDNF source. The interaction between β-actin mRNA and ZBP-1, a zipcode-binding protein that has previously been shown to regulate β-actin mRNA translocation, as well as asymmetric Src activity, which regulates β-actin translation by phosphorylating ZBP-1, appear to be critical for growth cone turning in response to BDNF or a local increase in intracellular Ca2+ — a known mediator of BDNF-induced growth cone turning.
In agreement with these findings, Christine Holt and colleagues show that netrin-1-induced attractive turning involves the translocation of β-actin mRNA and ZBP-1 into filopodia and asymmetric activation of the translation initiation regulator 4EBP, resulting in localized β-actin synthesis. Morpholino-mediated inhibition of β-actin translation prevented asymmetric β-actin accumulation and blocked netrin-1 induced growth cone turning. However, it did not block growth cone collapse or turning in response to repulsive cues, suggesting that asymmetric β-actin mRNA translation is specific for attractive guidance cues. While this model predicts that attraction and repulsion are the result of different pathways, Zheng and colleagues suggest that they are related processes due to the reversed asymmetry of β-actin levels in response to BDNF under repulsive conditions (PKA inhibition).
These papers highlight the importance of spatial regulation of cytoskeletal protein synthesis in axon guidance and suggest that Src and 4EBP regulate β-actin translation locally. It will be interesting to see whether other axon guidance cues also induce asymmetric actin synthesis and the local translation of cytoskeletal regulators such as RhoGTPases, which could also influence growth cone steering.
Monica Hoyos Flight Cell Migration Gateway
article Jiaqi Yao et al. An essential role for β-actin mRNA localization and translation in Ca2+-dependent growth cone guidance Nature Neuroscience 9, 1265 - 1273 (2006) Full text | PDF | Subscribe to Nature Neuroscience
article Kin-Mei Leung et al. Asymmetrical β-actin mRNA translation in growth cones mediates attractive turning to netrin-1 Nature Neuroscience 9, 1247 - 1256 (2006) Full text | PDF | Subscribe to Nature Neuroscience
Stem cell fate: What a stem cell Wnts
Activation of the Wnt signaling pathway in vivo leads to a loss of hematopoietic stem cell (HSC) differentiation and self-renewal potential.
Wnt signaling is well known for its involvement in development and pathogenesis. Previous studies have suggested that Wnt signaling plays a role in the renewal of the HSC population in the hematopoietic system, but the intracellular consequences resulting from the activation of the Wnt receptor Frizzled remain unclear. Binding of Wnt to Frizzled stabilizes β-catenin, which regulates expression of cell fate genes in a complex with Tcf-LEF transcription factors. Studies by Scheller et al. and by Kirstetter et al. in Nature Immunology now show that transgenic expression of a stable form of β-catenin blocks differentiation of murine HSCs.
To elucidate the consequences of conditionally overexpressed wild type β-catenin in mice, both groups designed a conditional expression system without retroviral or oncogenic components. In both studies, FACS analysis indicated that the differentiation of HSCs was arrested at early blood cell progenitor stages, leading to fatal anemia in the transgenic mice. Furthermore, failure of bone marrow engraftment by transformed HSCs in nude mice showed that the renewal potential of the HSCs was disrupted by β-catenin overexpression.
Using real-time polymerase chain reaction analysis, Kirstetter et al. discovered that the genes responsible for HSC population expansion (Cdkn1a, Hoxb4, and Bmi1) and myeloid progenitor formation (Sfp1) were significantly downregulated. However, the block of erythroid differentiation was due to ectopic PU.1 expression causing GATA-1 downregulation.
Scheller et al. in turn showed that transgenic HSCs had increased levels of cyclins E1 and E2 and reduced expression of the CDK inhibitory kinase p21kip/waf, which is essential for maintaining resting HSCs. Thus, the presence of β-catenin enables HSCs to enter the cell cycle but depletes the stem cell reservoir, resulting in a loss of long-term engraftment capacity.
The differentiation blockage of transgenic HSCs shown in both studies is similar to the phenotype of leukemic cells, yet the exhaustion rather than the renewal of the HSC population indicates that Wnt signaling is not sufficient for leukemic transformation. As components of the Wnt pathway are upregulated in several types of leukemia, the results of these two studies may allow targeting leukemic cells without disturbing HSCs by altering the Wnt pathway. Future studies should elucidate more precisely the strength and duration of canonical Wnt signaling required for normal HSC functioning.
Mirko von Elstermann Functional Glycomics Gateway
article Kirstetter, P. et al. Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block. Nature Immunology7, 1048 - 1056 (2006) Full text | PDF | Subscribe to Nature Immunology
article Scheller, M. et al. Hematopoietic stem cell and multilineage defects generated by constitutive β-catenin activation. Nature Immunology7, 1037 - 1047 (2006) Full text | PDF | Subscribe to Nature Immunology
news and views Trowbridge, J. J., Moon, R. T., Bhatia, M. Hematopoietic stem cell biology: too much of a Wnt thing. Nature Immunology7, 1021 - 1023 (2006) Full text | PDF | Subscribe to Nature Immunology
