ULK1/2 Constitute a Bifurcate Node Controlling Glucose Metabolic Fluxes in Addition to Autophagy.

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.

ProNodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm.

The secreted glycoprotein sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a crucial role in induction and patterning of the ventral forebrain. Currently little is known about how Shh is regulated in prechordal tissue. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, and is governed by unprocessed Nodal. Exposure of prechordal mesoderm microcultures to Nodal-conditioned medium, the Nodal inhibitor CerS, or to an ALK4/5/7 inhibitor reveals that Nodal is required to maintain both Shh and Gsc expression, but whereas Gsc is largely maintained through canonical signalling, Nodal signals through a non-canonical route to maintain Shh. Further, Shh expression can be maintained by a recombinant Nodal cleavage mutant, proNodal, but not by purified mature Nodal. A number of lines of evidence suggest that proNodal acts via FGFR3. ProNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Further, prechordal mesoderm cells in which Fgfr3 expression is reduced by Fgfr3 siRNA fail to bind to proNodal. Finally, targeted electroporation of Fgfr3 siRNA to prechordal mesoderm in vivo results in premature Shh downregulation without affecting Gsc. We report an inverse correlation between proNodal-FGFR3 signalling and pSmad1/5/8, and show that proNodal-FGFR3 signalling antagonises BMP-mediated pSmad1/5/8 signalling, which is poised to downregulate Shh. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and that local BMPs rapidly silence Shh once endogenous Nodal-FGFR3 signalling is downregulated.

Characterization of Cep85 - a new antagonist of Nek2A that is involved in the regulation of centrosome disjunction.

Nek2 has been implicated in centrosome disjunction at the onset of mitosis to promote bipolar spindle formation, and hyperactivation of Nek2 leads to the premature centrosome separation. Its activity, therefore, needs to be strictly regulated. In this study, we report that Cep85, an uncharacterized centrosomal protein, acts as a binding partner of Nek2A. It colocalizes with isoform A of Nek2 (Nek2A) at centrosomes and forms a granule meshwork enveloping the proximal ends of centrioles. Opposite to the effects of Nek2A, overexpression of Cep85 in conjunction with inhibition of the motor protein Eg5 (also known as KIF11) leads to the failure of centrosome disjunction. By contrast, depletion of Cep85 results in the precocious centrosome separation. We also define the Nek2A binding and centrosome localization domains within Cep85. Although the Nek2A-binding domain alone is sufficient to inhibit Nek2A kinase activity in vitro, both domains are indispensable for full suppression of centrosome disjunction in cells. Thus, we propose that Cep85 is a bona fide Nek2A-binding partner that surrounds the proximal ends of centrioles where it cooperates with PP1γ (also known as PPP1CC) to antagonize Nek2A activity in order to maintain the centrosome integrity in interphase in mammalian cells.

Cep85 Relays Plk1 Activity to Phosphorylated Nek2A for Its Timely Activation in Centrosome Disjunction.

Timely centrosome separation is critical for accurate chromosome separation. It is initiated by Nek2A at the onset of mitosis, but the mechanism for the strict requirement of phosphorylated Nek2A for its own activation remains unclear. In this study, we have found that Plk1 interacts with Cep85 and forms a ternary complex with Cep85-Nek2A. Nek2A binding, but not its kinase activity, is pre-required for Cep85 to be phosphorylated by Plk1. Nek2A-dependent Cep85 phosphorylation, in turn, leads to the dissociation of phosphorylated Cep85 exclusively from phospho-Nek2A, thereby increasing the freed phospho-Nek2A activity. Both kinases are also required for phosphorylating endogenous Cep85 in cells, and timely phosphorylation of Cep85 and Nek2A is crucial for initiating centrosome disjunction at G2/M. Overall, our study has uncovered a previously unrecognized role of Plk1 and Nek2A and identified Cep85 as a missing piece directly relaying Plk1 activity to Nek2A for its activation in centrosome disjunction.

Two modes by which Lefty proteins inhibit nodal signaling.

During vertebrate embryogenesis, members of the Lefty subclass of Transforming Growth Factor-beta (TGFbeta) proteins act as extracellular antagonists of the signaling pathway for Nodal, a TGFbeta-related ligand essential for mesendoderm formation and left-right patterning. Genetic and biochemical analyses have shown that Nodal signaling is mediated by activin receptors but also requires EGF-CFC coreceptors, such as mammalian Cripto or Cryptic. Misexpression experiments in zebrafish and frogs have suggested that Lefty proteins can act as long-range inhibitors for Nodal, possibly through competition for binding to activin receptors. Here we demonstrate two distinct and unexpected mechanisms by which Lefty proteins can antagonize Nodal activity. In particular, using a novel assay for Lefty activity in mammalian cell culture, we find that Lefty can inhibit signaling by Nodal but not by Activin or TGFbeta1, which are EGF-CFC independent. We show that Lefty can interact with Nodal in solution and thereby block Nodal from binding to activin receptors. Furthermore, Lefty can also interact with EGF-CFC proteins and prevent their ability to form part of a Nodal receptor complex. Our results provide mechanistic insights into how Lefty proteins can achieve efficient and stringent regulation of a potent signaling factor.

The Vg1-related protein Gdf3 acts in a Nodal signaling pathway in the pre-gastrulation mouse embryo.

The formation of the anterior visceral endoderm (AVE) in the pre-gastrulation mouse embryo represents a crucial event in patterning of the anterior-posterior axis. Here, we show that the transforming growth factor beta (Tgfbeta) family member Gdf3 (growth-differentiation factor 3), a close relative of Xenopus Vg1, resembles the Tgfbeta ligand Nodal in both its signaling activity and its role in AVE formation in vivo. Thus, in cell culture, Gdf3 signaling requires the EGF-CFC co-receptor Cripto and can be inhibited by Lefty antagonists. In Xenopus embryos, Gdf3 misexpression results in secondary axis formation, and induces morphogenetic elongation and mesendoderm formation in animal caps. In mouse embryos, Gdf3 is expressed in the inner cell mass and epiblast, and null mutants frequently exhibit abnormal formation or positioning of the AVE. This phenotype correlates with defects in mesoderm and definitive endoderm formation, as well as abnormal Nodal expression levels. Our findings indicate that Gdf3 acts in a Nodal-like signaling pathway in pre-gastrulation development, and provide evidence for the functional conservation of Vg1 activity in mice.

Inhibition of excess nodal signaling during mouse gastrulation by the transcriptional corepressor DRAP1.

The formation and patterning of mesoderm during mammalian gastrulation require the activity of Nodal, a secreted mesoderm-inducing factor of the transforming growth factor-beta (TGF-beta) family. Here we show that the transcriptional corepressor DRAP1 has a very specific role in regulation of Nodal activity during mouse embryogenesis. We find that loss of Drap1 leads to severe gastrulation defects that are consistent with increased expression of Nodal and can be partially suppressed by Nodal heterozygosity. Biochemical studies indicate that DRAP1 interacts with and inhibits DNA binding by the winged-helix transcription factor FoxH1 (FAST), a critical component of a positive feedback loop for Nodal activity. We propose that DRAP1 limits the spread of a morphogenetic signal by down-modulating the response to the Nodal autoregulatory loop.