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.
New components of the Fat tumor suppressor pathway reveal unexpected links to other growth-regulating pathways.
Activation of the protocadherin Fat at the cell membrane triggers a signal transduction cascade that regulates tissue polarity and growth during development in Drosophila. Fat induces the expression of genes involved in wing development such as wingless (wg), four-jointed (fj) and serrate (ser) but the mechanism is unclear. In Nature Genetics, Kenneth Irvine and colleagues find that discs overgrown (dco) and warts (wts) are novel components of the Fat signaling pathway, revealing unexpected links to the Hippo tumor suppressor pathway.
The authors show that the characteristic broadening of the wg expression domain in the proximal wing of Fat mutants is also observed in the mutants of the tumor suppressor genes dco, expanded (ex), mob as tumor suppressor (matts), salvador (sav), Hippo and wts. These tumor genes share transcriptional targets with Fat, indicating that they might function in the same signaling cascade. Epistasis experiments revealed that dco acts upstream of Fat, and confirm that Fat acts upstream of dachs (the only previously identified downstream component of the Fat tumor suppressor pathway). Furthermore, dachs acts upstream of merlin and ex, two components of the Hippo signaling pathway which are known to act upstream of wts. These findings are consistent with two other papers in Current Biology1, 2 that propose that Fat is actually the receptor of the Hippo signaling pathway.
Interestingly, Irvine and colleagues show that Fat and Hippo exert different effects on wts. Loss of Fat function leads to a reduction in the levels of wts protein, whereas Hippo changes wts activity by regulating its phosphorylation. They suggest that wts functions as an integrator of distinct growth signals that can be transmitted by both the Fat pathway and the Hippo pathway.
Finally, they show that wts co-immunoprecipitates with dachs, suggesting that dachs contributes to reduce the levels of wts by acting as a scaffold for a wts degradation complex, and that the transcriptional co-activator and downstream component of the Hippo pathway yorkie (yki) is involved in the induction of Fat-wts target gene expression. Future studies will uncover more details of the cross-regulation between the Hippo and Fat tumor suppressor pathways, and show whether the mammalian homologues of wts (Lats1 and Lats2) also function as convergence points for conserved growth-regulating pathways.
Monica Hoyos Flight Cell Migration Gateway
article Eunjoo Cho, Yongqiang Feng, Cordelia Rausko, Sushmita Maitra, Rick Fehon & Kenneth D Irvine Delineation of a Fat tumor suppressor pathway Nature Genetics, doi:10.1038/ng1887 Full text | PDF | Subscribe to Nature Genetics
References
Elizabeth Silva et al. The Tumor-Suppressor Gene Fat Controls Tissue Growth Upstream of Expanded in the Hippo Signaling Pathway. Current Biology, in press. | Abstract |
Maria Willecke et al. The Fat Cadherin Acts through the Hippo Tumor-Suppressor Pathway to Regulate Tissue Size. Current Biology, in press. | Abstract |
β-cell differentiation: NFATuated with calcineurin
The nuclear re-localization of NFAT protein complexes increases β-cell proliferation and insulin production.
Pancreatic β-cells are the sole source of insulin in vertebrates. It has recently been shown that these cells grow facultatively and that their growth reflects changes in insulin demand associated with physiological changes such as pregnancy, ageing and obesity. Signaling via the transcription factor calcineurin/nuclear factor of activated T-cells (NFAT) is implicated in insulin regulation, although the precise molecular mechanism has remained elusive. A study by Heit et al. in Nature reveals that calcineurin-mediated NFAT nuclear localization regulates β-cell proliferation.
Physiological or developmental cues in lymphocytes, myocytes and neurons trigger increases in intracellular Ca2+ leading to activation of the serine/ threonine phosphatase calcineurin. Calcineurin induces rapid nuclear localization of NFAT transcription complexes. It was previously shown that insulin and glucose increase intracellular Ca2+ concentrations and that proteins of NFAT complexes are activated in β-cells. These findings led the authors to investigate the specific role of NFAT in β-cell differentiation. Mice with genetic deletion of calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1) (Cnb1KO) accumulated less NFATc1 in β-cells, accompanied by severe hyperglycemia and overt diabetes mellitus onset by week 10. Insulin production was reduced by 85% and β-cell mass was also reduced by 50%.
β-cells of Cnb1KO mice have reduced mRNA and protein expression of the glucose transporter Glut2, β-cell-specific transcription factors and cell cycle regulators. Taken together, these results suggest that loss of Cnb1 leads to attenuated insulin synthesis due to reduced proliferation of β-cells.
Chromatin immunoprecipitation (ChIP) uncovered NFATc1 binding sites in the promoters of the depressed genes, including Ins1, Hnf4a, Gck, Glut2 and Cdk4. Binding was abolished after treatment with cyclosporine A, a calcineurin inhibitor. A calcineurin-independent constitutively nuclear NFAT mutant restored mRNA levels of β-cell specific genes such as Ins1 as well as serum insulin levels and pancreatic mass in mice. Thus, the authors conclude that the nuclear localization of the NFAT protein complex is necessary and sufficient to increase the expression of genes regulating β-cell proliferation and function. NFAT is therefore a promising therapeutic target not just for the treatment of diabetes, but also in cases of disordered β-cell growth, such as insulinomas or nesidioblastosis.
Clare Garvey Signaling Gateway
letter Jeremy J. Heit, Åsa A. Apelqvist, Xueying Gu, Monte M. Winslow, Joel R. Neilson, Gerald R. Crabtree, and Seung K. Kim Calcineurin/NFAT signalling regulates pancreatic β-cell growth and function Nature, 443, 345-349 (21 September 2006) | doi:10.1038/nature05097 Full text | PDF | Subscribe to Nature
Ion channels: CRAC-king store-operated calcium entry
Conserved residues of the transmembrane protein Orai determine the ion selectivity of CRAC channels.
Ca2+ is a universal second messenger controlling a wide variety of cellular reactions and adaptive responses. Ca2+ release-activated Ca2+ (CRAC) channels mediate Ca2+ influx across the plasma membrane when intracellular stores are depleted. Despite the fact that the pharmacological and physiological properties of CRAC channels have been described in detail, their molecular identity has remained elusive. The transmembrane proteins Stim (stromal interaction molecule) and Orai have recently been shown to activate CRAC channels, but two new studies in Nature, as well as a previous study in Nature Cell Biology1, indicate that Orai is more than a regulatory component. Point mutations of key acidic residues alter the ion selectivity of CRAC channels, strongly suggesting that Orai is actually the pore-forming subunit.
In one Nature study, Yeromin et al. show that the interaction between Stim and Orai is important for CRAC channel activation following thapsigargin-induced Ca2+ store depletion in S2 cells. More importantly, functional analyses of Orai variants with point mutations in the S1-S2 region, the region most conserved between Drosophila and human Orai, reveal that glutamate 180 is a crucial residue for CRAC channel ion selectivity. A glutamate to aspartate mutation at this position transforms CRAC channels from being Ca2+ selective with inward rectification to being selective for monovalent cations and outwardly rectifying. On the other hand, a glutamate to alanine mutation prevented ion conduction altogether suggesting that Orai is a constitutive CRAC channel subunit.
These findings are in agreement with the study by Prakriya et al. that examines the function of recombinant Orai proteins in T cells and fibroblasts. In this paper, the authors identify two conserved acidic residues in human Orai that may be involved in the regulation of Ca2+ entry — glutamate 106, which corresponds to glutamate 180 in the Drosophila protein, and glutamate 190. Mutating glutamate 106 to aspartate, or glutamate 190 to glutamine, not only decreased CRAC channel sensitivity to Ca2+, it also reduced the blockade of Na+ currents by Ca2+ and increased channel permeability to Cs+.
Together these studies strongly support the idea that Orai is not just an activator of CRAC channels, it is a key subunit regulating ion selectivity. It is likely that E106/E180 mediates the interaction of Ca2+ with the channel and determines pore size. Further understanding the architecture of CRAC channels will aid efforts towards manipulating them pharmacologically.
Monica Hoyos Flight Cell Migration Gateway
letter Andriy V. Yeromin, Shenyuan L. Zhang, Weihua Jiang, Ying Yu, Olga Safrina and Michael D. Cahalan Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai Nature, 443, 226-229 (2006) | doi:10.1038/nature05108 Full text | PDF | Subscribe to Nature
letter Murali Prakriya, Stefan Feske, Yousang Gwack, Sonal Srikanth, Anjana Rao and Patrick G. Hogan Orai1 is an essential pore subunit of the CRAC channel Nature, 443, 230-233 (2006) | doi:10.1038/nature05122 Full text | PDF | Subscribe to Nature
References
Peinelt, Christine et al. Amplification of CRAC current by STIM1 and CRACM1 (Orai1) Nature Cell Biology8, 771–773 (2006) | Article |
The Rho GTPase cdc42 maintains the fate of ventricular zone progenitors and adherens junctions in mammalian neuronal development.
In the mammalian brain, neurogenesis continues into adulthood and is dependent on the asymmetric division of neural progenitor cells. Apically located neuroepithelia progenitors in the ventricular zone (VZ) give rise to further progenitor cells as well as post-mitotic neurons. On the other hand, basally located progenitors in the sub ventricular zone (SVZ) divide to generate two neurons. The molecular events governing the adoption of these different fates remain poorly understood. Now, Silvia Cappello et al. in Nature Neuroscience reveal a novel role for the Rho GTPase cdc42 in the maintenance of the self-renewing fate of VZ progenitor cells.
Both SVZ and VZ progenitors exhibit apical-basal polarity; however, SVZ cells differ from VZ cells in the basal position of their mitosis. One other notable difference between VZ and SVZ cells is the expression of the Par complex in VZ progenitors. This complex is a well known regulator of apical polarity, adherens junction formation and asymmetric division and it is known to be regulated by cdc42.
The authors show that cdc42 is expressed in VZ progenitors and also in basally located postmitotic neurons, but not in basally located progenitors. Deletion of the second exon of cdc42 in mice resulted in a reduced survival rate accompanied by a severely disorganized cortex. The authors investigated whether the loss of cdc42 affected both progenitor subtypes; in cdc42-deficient cortex, many basal mitoses were observed where apical mitoses should predominate. An increase in neuron number was also seen, and analysis of SZ or SVZ-specific transcription factors revealed that cdc42-deficient apical progenitors had adopted a basal progenitor fate. These results are consistent with an increase in basal progenitor numbers.
The difference in location of progenitor mitoses and the known role for cdc42 in centrosome positioning and cell cycle length led the authors to investigate the effect of loss of cdc42 on these processes. Both cell cycle length and spindle orientation in dividing progenitor cells were unaffected. However, the nuclei in cdc42-deficient apical progenitors showed reduced apical migration. Furthermore, cdc42-deficient apical patches are devoid of adherens junctions and show a loss of Par complex proteins and aPKC. Thus, the GTPase cdc42 is required in mammalian neurogenesis for maintaining adherens junctions coupling and the VZ progenitor fate.
Clare Garvey Signaling Gateway
article Silvia Cappello, Alessio Attardo, Xunwei Wu, Takuji Iwasato, Shigeyoshi Itohara, Michaela Wilsch-Bräuninger, Hanna M Eilken, Michael A Rieger, Timm T Schroeder, Wieland B Huttner, Cord Brakebusch & Magdalena Götz The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface Nature Neuroscience, 9, 1099-1107 (2006) | doi:10.1038/nn1744 Full text | PDF | Subscribe to Nature Neuroscience
EGFR Signaling: Keeping its Cool
Phosphorylation of Cool-1 prevents EGFR endocytosis and is essential for v-Src-induced transformation.
Cool-1 (cloned-out of library; also known as β-Pix) prevents epidermal growth factor receptor (EGFR) degradation by binding to Cdc42 and sequestering the ubiquitin ligase Cbl. Further examination of the mechanism through which Cool-1 affects EGFR signaling has uncovered an important role for Cool-1 as a mediator of v-Src-induced cell transformation and tumor growth.
In Nature Cell Biology, Richard Cerione and colleagues now show that both Cdc42 and Cool-1 are required for EGF-induced activation of ERK kinase. EGF signaling through Src and FAK induces transient phosphorylation of Cool-1 on Tyr442, which turns on its guanine nucleotide exchange factor (GEF) activity, leading to Cdc42 activation. Moreover, both Cbl and active Cdc42 preferentially bind to phosphorylated Cool-1. EGFRs also bind to phosphorylated Cool-1 forming a ternary complex with Cbl, but not Cdc42. As EGFR was not detected in Cdc42-Cool-1-Cbl complexes, the authors suggest that active Cdc42 and EGFRs compete for Cool-1 and Cbl binding.
The amount of EGFRs on the cell surface was dramatically increased when Cool-1 was overexpressed and constitutively phosphorylated by overexpressed v-Src kinase. This effect was abolished when v-Src was expressed with either Cool-1 RNAi or a phosphorylation defective Cool-1 mutant. These findings indicate that phosphorylated Cool-1 blocks EGFR endocytosis, although the role of the Cdc42-Cool-1-Cbl ternary complexes remains unclear. Interestingly, reducing the levels of Cool-1 inhibited v-Src-induced cell growth and proliferation, whereas coexpression of Cool-1 with v-Src enhanced the formation of large cellular aggregates and increased tumor formation in mice.
By identifying these two complexes, the authors shed new light on Cool-1's role in EGFR signaling. They propose that Cdc42-Cool-1-Cbl complexes sequester Cbl to prevent EGFR degradation. On the other hand, Cool-1's association with EGFR blocks receptor endocytosis, potentially by preventing Cbl from binding the endocytic machinery. In both cases EGFR signaling is enhanced, but, as this study points out, maintaining the balance between these complexes is essential for preventing v-Src induced transformation.
Monica Hoyos Flight Cell Migration Gateway
article Qiyu Feng, Dan Baird, Xu Peng, Jianbin Wang, Thi Ly, Jun-Lin Guan & Richard A. Cerione Cool-1 functions as an essential regulatory node for EGFreceptor- and Src-mediated cell growth Nature Cell Biology, (6 August 2006) | doi:10.1038/ncb1453 Full text | PDF | Subscribe to Nature Cell Biology
