Phytochrome signalling is mediated through nucleoside diphosphate kinase 2.

Because plants are sessile, they have developed intricate strategies to adapt to changing environmental variables, including light. Their growth and development, from germination to flowering, is critically influenced by light, particularly at red (660 nm) and far-red (730 nm) wavelengths. Higher plants perceive red and far-red light by means of specific light sensors called phytochromes(A-E). However, very little is known about how light signals are transduced to elicit responses in plants. Here we report that nucleoside diphosphate kinase 2 (NDPK2) is an upstream component in the phytochrome signalling pathway in the plant Arabidopsis thaliana. In animal and human cells, NDPK acts as a tumour suppressor. We show that recombinant NDPK2 in Arabidopsis preferentially binds to the red-light-activated form of phytochrome in vitro and that this interaction increases the activity of recombinant NDPK2. Furthermore, a mutant lacking NDPK2 showed a partial defect in responses to both red and farred light, including cotyledon opening and greening. These results indicate that NDPK2 is a positive signalling component of the phytochrome-mediated light-signal-transduction pathway in Arabidopsis.     

Platelet-derived growth factor-alpha receptor activation is required for human cytomegalovirus infection

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus that can cause life-threatening disease in the fetus and the immunocompromised host. Upon attachment to the cell, the virus induces robust inflammatory, interferon- and growth-factor-like signalling. The mechanisms facilitating viral entry and gene expression are not clearly understood. Here we show that platelet-derived growth factor-alpha receptor (PDGFR-alpha) is specifically phosphorylated by both laboratory and clinical isolates of HCMV in various human cell types, resulting in activation of the phosphoinositide-3-kinase (PI(3)K) signalling pathway. Upon stimulation by HCMV, tyrosine-phosphorylated PDGFR-alpha associated with the p85 regulatory subunit of PI(3)K and induced protein kinase B (also known as Akt) phosphorylation, similar to the genuine ligand, PDGF-AA. Cells in which PDGFR-alpha was genetically deleted or functionally blocked were non-permissive to HCMV entry, viral gene expression or infectious virus production. Re-introducing human PDGFRA gene into knockout cells restored susceptibility to viral entry and essential viral gene expression. Blockade of receptor function with a humanized PDGFR-alpha blocking antibody (IMC-3G3) or targeted inhibition of its kinase activity with a small molecule (Gleevec) completely inhibited HCMV viral internalization and gene expression in human epithelial, endothelial and fibroblast cells. Viral entry in cells harbouring endogenous PDGFR-alpha was competitively inhibited by pretreatment with PDGF-AA. We further demonstrate that HCMV glycoprotein B directly interacts with PDGFR-alpha, resulting in receptor tyrosine phosphorylation, and that glycoprotein B neutralizing antibodies inhibit HCMV-induced PDGFR-alpha phosphorylation. Taken together, these data indicate that PDGFR-alpha is a critical receptor required for HCMV infection, and thus a target for novel anti-viral therapies.     

Robustness of the BMP morphogen gradient in Drosophila embryonic patterning

Developmental patterning relies on morphogen gradients, which generally involve feedback loops to buffer against perturbations caused by fluctuations in gene dosage and expression. Although many gene components involved in such feedback loops have been identified, how they work together to generate a robust pattern remains unclear. Here we study the network of extracellular proteins that patterns the dorsal region of the Drosophila embryo by establishing a graded activation of the bone morphogenic protein (BMP) pathway. We find that the BMP activation gradient itself is robust to changes in gene dosage. Computational search for networks that support robustness shows that transport of the BMP class ligands (Scw and Dpp) into the dorsal midline by the BMP inhibitor Sog is the key event in this patterning process. The mechanism underlying robustness relies on the ability to store an excess of signalling molecules in a restricted spatial domain where Sog is largely absent. It requires extensive diffusion of the BMP-Sog complexes, coupled with restricted diffusion of the free ligands. We show experimentally that Dpp is widely diffusible in the presence of Sog but tightly localized in its absence, thus validating a central prediction of our theoretical study.     

Repressor activity of Headless/Tcf3 is essential for vertebrate head formation

The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wnt antagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wnt signalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. Here we describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. We provide genetic evidence that a component of the Wnt signalling pathway is essential in vertebrate head formation and patterning.     

The MAPK kinase Pek1 acts as a phosphorylation-dependent molecular switch.

The mitogen-activated protein kinase (MAPK) pathway is a highly conserved eukaryotic signalling cascade that converts extracellular signals into various outputs, such as cell growth and differentiation. MAPK is phosphorylated and activated by a specific MAPK kinase (MAPKK): MAPKK is therefore considered to be an activating regulator of MAPK. Pmk1 is a MAPK that regulates cell integrity and which, with calcineurin phosphatase, antagonizes chloride homeostasis in fission yeast. We have now identified Pek1, a MAPKK for Pmk1 MAPK. We show here that Pek1, in its unphosphorylated form, acts as a potent negative regulator of Pmk1 MAPK signalling. Mkh1, an upstream MAPKK kinase (MAPKKK), converts Pek1 from being an inhibitor to an activator. Our results indicate that Pek1 has a dual stimulatory and inhibitory function which depends on its phosphorylation state. This switch-like mechanism could contribute to the all-or-none physiological response mediated by the MAPK signalling pathway.     

Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck

Rac signalling to actin -- a pathway that is thought to be mediated by the protein Scar/WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein -- has a principal role in cell motility. In an analogous pathway, direct interaction of Cdc42 with the related protein N-WASP stimulates actin polymerization. For the Rac-WAVE pathway, no such direct interaction has been identified. Here we report a mechanism by which Rac and the adapter protein Nck activate actin nucleation through WAVE1. WAVE1 exists in a heterotetrameric complex that includes orthologues of human PIR121 (p53-inducible messenger RNA with a relative molecular mass (M(r)) of 140,000), Nap125 (NCK-associated protein with an M(r) of 125,000) and HSPC300. Whereas recombinant WAVE1 is constitutively active, the WAVE1 complex is inactive. We therefore propose that Rac1 and Nck cause dissociation of the WAVE1 complex, which releases active WAVE1-HSPC300 and leads to actin nucleation.     

Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein.

Oocytes arrested in the G2 phase of the cell cycle contain a p34cdc2/cyclin B complex which is kept in an inactive form by phosphorylation of its p34cdc2 subunit on tyrosine, threonine and perhaps serine residues. The phosphatase(s) involved in p34cdc2 dephosphorylation is unknown, but the product of the fission yeast cdc25+ gene, and its homologues in budding yeast and Drosophila are probably positive regulators of the transition from G2 to M phase. We have purified the inactive p34cdc2/cyclin B complex from G2-arrested starfish oocytes. Addition of the purified bacterially expressed product of the human homologue of the fission yeast cdc25+ gene (p54CDC25H) triggers p34cdc2 dephosphorylation and activates H1 histone kinase activity in this preparation. We propose that the cdc25+ gene product directly activates the p34cdc2-cyclin B complex.     

Notch activity acts as a sensor for extracellular calcium during vertebrate left-right determination

During vertebrate embryo development, the breaking of the initial bilateral symmetry is translated into asymmetric gene expression around the node and/or in the lateral plate mesoderm. The earliest conserved feature of this asymmetric gene expression cascade is the left-sided expression of Nodal, which depends on the activity of the Notch signalling pathway. Here we present a mathematical model describing the dynamics of the Notch signalling pathway during chick embryo gastrulation, which reveals a complex and highly robust genetic network that locally activates Notch on the left side of Hensen's node. We identify the source of the asymmetric activation of Notch as a transient accumulation of extracellular calcium, which in turn depends on left-right differences in H+/K+-ATPase activity. Our results uncover a mechanism by which the Notch signalling pathway translates asymmetry in epigenetic factors into asymmetric gene expression around the node.     

Bacteriophytochrome controls photosystem synthesis in anoxygenic bacteria

Plants use a set of light sensors to control their growth and development in response to changes in ambient light. In particular, phytochromes exert their regulatory activity by switching between a biologically inactive red-light-absorbing form (Pr) and an active far-red-light absorbing form (Pfr). Recently, biochemical and genetic studies have demonstrated the occurrence of phytochrome-like proteins in photosynthetic and non-photosynthetic bacteria--but little is known about their functions. Here we report the discovery of a bacteriophytochrome located downstream from the photosynthesis gene cluster in a Bradyrhizobium strain symbiont of Aeschynomene. The synthesis of the complete photosynthetic apparatus is totally under the control of this bacteriophytochrome. A similar behaviour is observed for the closely related species Rhodopseudomonas palustris, but not for the more distant anoxygenic photosynthetic bacteria of the genus Rhodobacter, Rubrivivax or Rhodospirillum. Unlike other (bacterio)phytochromes, the carboxy-terminal domain of this bacteriophytochrome contains no histidine kinase features. This suggests a light signalling pathway involving direct protein-protein interaction with no phosphorelay cascade. This specific mechanism of regulation may represent an important ecological adaptation to optimize the plant-bacteria interaction.     

Wnt proteins are lipid-modified and can act as stem cell growth factors

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Here we have isolated active Wnt molecules, including the product of the mouse Wnt3a gene. By mass spectrometry, we found the proteins to be palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of haematopoietic stem cells, signifying its potential use in tissue engineering.