Neuronal stem cells: Less Notch, more neurons

Epidermal growth factor-like domain 7 (EGFL7) antagonizes Notch signaling to limit neural stem cell self renewal and promote neurogenesis.

Notch signaling in neural stem cells (NSCs) stimulates proliferation and self renewal. The Notch ligands Jagged1 and Jagged2 bind to its extracellular domain, promoting cleavage of the Notch intracellular domain (NICD), which translocates to the nucleus to modulate transcription. In Nature Cell Biology, Ivan Dikic and colleagues now show that epidermal growth factor-like domain 7 (EGFL7) binds to and antagonizes Notch, inhibiting NSC renewal and promoting neurogenesis.

A yeast two-hybrid screen identified an interaction between EGFL7 and the ligand-binding EGF-like extracellular domains of Notch family members. EGFL7 effectively competed with Jagged1 or Jagged2 for access to Notch1, and EGFL7 binding reduced Jagged-induced generation of the NICD. Jagged1–Notch1 signaling is known to be essential for NSC self renewal, and Dikic and colleagues found that exogenous EGFL7 reduced NSC proliferation and transcription of Notch1 target genes, similar to the effect of Notch pathway inhibitors. Exogenous NICD reversed the EGFL7-mediated decrease in NSC self renewal. In contrast, EGFL7 antisense or small interfering (si)RNA enhanced transcription and NSC renewal. These data suggest that EGFL7 antagonizes Notch signaling by interfering with ligand binding and subsequent proteolysis.

In addition to regulating NSC proliferation, Notch signaling is thought to favor the differentiation of neural precursors into astrocytes. Intriguingly, similar to the Notch inhibitor DAPT, EGFL7 stimulated the differentiation of cultured neurospheres into neurons and oligodendrites, but not astrocytes. However, EGFL7 antisense favored the astrocyte lineage. EGFL7 mRNA expression was 80-fold higher in differentiated neurospheres than in undifferentiated, providing further support that EGFL7 promotes NSC differentiation.

EGFL7 is known to modulate vascular biology and angiogenesis. This study reveals a novel neurobiological role for EGFL7 in antagonizing Notch signaling to limit NSC self renewal and promote neurogenesis. It will be important to determine how EGFL7 is regulated, and to explore any potential links between neural differentiation and angiogenesis in light of this new point of conversion.

Emily J. Chenette
Signaling Gateway

Reference:
Schmidt, M. H. H. et al.
Epidermal growth factor-like domain 7 (EGFL7) modulates Notch signalling and affects neural stem cell renewal
Nature Cell Biology advance online publication, 7 June 2009 (DOI 10.1038/ncb1896)
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Infectious disease: Mycobacteria set up cAMP in macrophages

Phagocytosed Mycobacterium tuberculosis produces a burst of cAMP in macrophages that subverts the host immune response.

Mycobacterium tuberculosis, the casual agent of tuberculosis, is phagocytosed by macrophages, where it elicits a burst of cyclic AMP (cAMP) that interferes with phagosome maturation and host signaling. However, the mechanism by which M. tuberculosis stimulates cAMP production has not yet been identified. In Nature, William Bishai and colleagues now report that a bacterially expressed adenylyl cyclase produces the cAMP burst, which inhibits tumor necrosis factor-α (TNF-α) expression via the protein kinase A (PKA)-cAMP response-element-binding protein (CREB) pathway.

Brief exposure of macrophages to live M. tuberculosis stimulated a burst of intracellular cAMP, which induced PKA-mediated CREB phosphorylation. Surprisingly, pharmacological inhibition of macrophage adenylyl cyclase activity did not affect cAMP levels, suggesting an alternative source of cAMP production. Indeed, radiolabeling confirmed that bacterially derived cAMP was the source of the burst.

Mutation of the bacterial cell-wall-associated adenylyl cyclase Rv0386 impaired cAMP production, which was restored with exogenous Rv0386. Mutant Rv0386 also reduced both phospho-CREB levels and the secretion of TNF-α by macrophages, indicating that M. tuberculosis adenylyl cyclase influences host immune signaling through cAMP production. Indeed, M. tuberculosis expressing the mutant Rv0386 did not thrive in the lung tissue of wild-type mice, but were able to proliferate in TNF-α-deficient mice. Thus, M. tuberculosis-mediated cAMP production subverts host signaling to induce early TNF-α secretion, which is critical for its survival.

Bacteria have evolved a myriad of strategies for disrupting host cell signaling to permit uptake or sustained colonization. These data provide evidence that M. tuberculosis hijacks macrophage signaling pathways via direct production of the secondary signaling molecule cAMP. It will be important to determine whether bacterial adenylyl cyclase activity can be selectively blocked, as this strategy would represent an important advance in tuberculosis treatment.

Emily J. Chenette
Signaling Gateway

Reference:
Agarwal, N., Lamichhane, G., Gupta, R., Nolan, S. and Bishai, W. R.
Cyclic AMP intoxication of macrophages by a Mycobacterium tuberculosis adenylate cyclase
Nature advance online publication, 10 June 2009 (DOI 10.1038/nature08123)
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Kinases: An activating inhibitor?

ATP-competitive inhibitors induce phosphorylation priming of the protein kinases Akt and PKC, suggesting that nucleotide binding, rather than intrinsic catalytic activity, stimulates kinase activation.

Members of the AGC family of protein kinases, including Akt (also called protein kinase B (PKB)) and PKC, have important functions in normal and pathological cellular processes. Studies on their mode of activation have shown that 'priming' phosphorylation events in the kinase activation loop are required for AGC kinase activity. Now, two studies reveal that occupancy of the nucleotide-binding pocket by ATP or an ATP-competitive inhibitor induces phosphorylation priming of Akt and PKC independently of intrinsic kinase activity.

Full Akt activation requires three distinct upstream events: phosphatidylinositol-1,4,5-trisphosphate-dependent membrane translocation, phosphoinositide-dependent kinase 1 (PDK1)-mediated phosphorylation at Thr 308 and phosphorylation at Ser 473 by mammalian target of rapamycin complex 2 (mTORC2). Reporting in Nature Chemical Biology, Okuzumi et al. used a chemical-genetic approach to develop specific ATP-competitive Akt inhibitors. Intriguingly, the inhibition of Akt kinase activity by these inhibitors was accompanied by hyperphosphorylation at Thr 308 and Ser 473. Association of Akt with the membrane was necessary but not sufficient to induce hyperphosphorylation in response to inhibitors.

So, how do these inhibitors lead to phosphorylation? The finding that a kinase-dead mutant of Akt was still phosphorylated suggests that a property of inhibitor binding, rather than an extrinsic feedback loop, stimulates phosphorylation. Indeed, the inhibitors triggered Akt membrane translocation and phosphorylation by PDK1 and mTORC2 – all of the events required for full Akt activation. The authors speculate that occupancy of the ATP-binding pocket induces a conformational change in Akt that promotes membrane association and exposes Thr 308 and Ser 473 for phosphorylation.

In a separate study in Nature Structural and Molecular Biology, Cameron et al. found that kinase-inactive PKCε mutants that cannot coordinate ATP effectively are nonetheless phosphorylated in response to ATP-competitive inhibitors at three phosphorylation priming sites (Thr 566, Thr 710 and Ser 729). In addition, a catalytically inactive PKCε mutant that could coordinate ATP was phosphorylated independently of the inhibitors. Similar to the observations for Akt, these findings suggest that nucleotide pocket occupation, and not autophosphorylation or a feedback loop, might be sufficient to induce the phosphorylation of PKCε. Remarkably, activation-associated displacement of ATP from the nucleotide-binding pocket of a PKCε mutant that weakly binds ATP coincided with rapid dephosphorylation of all three PKCε priming sites. Furthermore, ATP-competitive inhibitors stimulated rephosphorylation of these sites, which suggests that nucleotide pocket occupancy dictates kinase activation. Similar observations were made for PKCα.

These data provide evidence that ATP binding to AGC kinases is an essential first step that stimulates their subsequent phosphorylation and activation. The activation of G proteins is regulated by guanine nucleotide-induced conformational changes, and it will be interesting to determine the extent to which adenosine nucleotides alter AGC kinase conformation and regulate their activation in a physiological setting. It will also be important to understand how ATP binding is regulated for AGC kinases, as well as the effect, if any, of priming phosphorylation events on upstream or downstream signaling pathways.

Emily J. Chenette
Signaling Gateway

References:
Okuzumi, T. et al.
Inhibitor hijacking of Akt activation
Nature Chemical Biology advance online publication, 24 May 2009 (DOI 10.1038/nchembio.183)
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Cameron, A. J. M. et al.
PKC maturation is promoted by nucleotide pocket occupation independently of intrinsic kinase activity
Nature Structural & Molecular Biology 16, 624-630 (2009)
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Frye, S. V. and Johnson, G. L.
Inhibitors paradoxically prime kinases
Nature Chemical Biology advance online publication, 24 May 2009 (DOI 10.1038/nchembio.f.11)
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Cancer: A tumor suppressor is DSCRibed on chromosome 21

The protein products of two genes on chromosome 21 — Dscr1 and Dyrk1a — suppress angiogenesis and inhibit tumor growth by antagonizing VEGF signaling.

Individuals with Down's syndrome display a lower incidence of many types of cancer, and the candidate genes on chromosome 21 that confer this protection from cancer are actively sought. In Nature, Sandra Ryeom and colleagues now report that two chromosome-21 genes, Dscr1 (Down's syndrome candidate region-1) and Dyrk1a (dual-specificity tyrosine phosphorylation-regulated kinase 1A), block tumor cell expansion by inhibiting angiogenesis.

In addition to a lower prevalence of solid tumors, individuals with Down's syndrome also have a reduced occurrence of angiogenesis-related diseases. Among the 231 trisomic genes in Down's syndrome individuals, the Dscr1 gene product has been shown to negatively regulate VEGF–calcineurin signaling and block VEGF-mediated angiogenesis. Ryeom and colleagues found that Dscr1 protein levels were elevated in Down's syndrome embryos and in a mouse model of Down's syndrome (Ts65D). The growth and vascularization of transplanted lung cancer and melanoma cells was diminished in Ts65D mice, and transgenic mice expressing an additional copy of Dscr1 showed a similar impairment in transplanted tumor cell growth and microvessel density. Ts65D mice that contained two, rather than three copies of Dscr1 also lost protection from tumorigenesis.

Endothelial cells from both Ts65D and transgenic mice displayed decreased proliferation in response to VEGF, an effect that could be attributed to misregulated VEGF–calcineurin–NFAT signaling, as Dscr1 transgenic mice expressed lower levels of the calcineurin-dependent, pro-angiogenic gene COX2. Intriguingly, another chromosome-21 gene, Dyrk1a, also negatively regulates NFAT-mediated transcription. Combined Dyrk1a upregulation and Dscr1 trisomy severely inhibited VEGF-stimulated endothelial cell proliferation. Therefore, the additional copies of Dscr1 and Dyrk1a appear to inhibit tumor growth by blocking the VEGF–calcineurin–NFAT axis in blood vessels.

This study links the overexpression of the chromosome-21 genes Dscr1 and Dyrk1a to the inhibition of angiogenesis and tumor suppression present in Down's syndrome individuals. As both these proteins antagonize VEGF signaling, it will be important to determine to what extent Dscr1 and Dyrk1a act synergistically to suppress angiogenesis.

Emily J. Chenette
Signaling Gateway

Reference:
Baek, K.-H., Zaslavsky, A., Lynch, R. C. et al.
Down's syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1
Nature advance online publication, 20 May 2009 (DOI 10.1038/nature08062)
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