A conserved role for Notch signaling in priming the cellular response to Shh through ciliary localisation of the key Shh transducer Smo.

Notochord-derived Sonic Hedgehog (Shh) is essential for dorsoventral patterning of the overlying neural tube. Increasing concentration and duration of Shh signal induces progenitors to acquire progressively more ventral fates. We show that Notch signalling augments the response of neuroepithelial cells to Shh, leading to the induction of higher expression levels of the Shh target gene Ptch1 and subsequently induction of more ventral cell fates. Furthermore, we demonstrate that activated Notch1 leads to pronounced accumulation of Smoothened (Smo) within primary cilia and elevated levels of full-length Gli3. Finally, we show that Notch activity promotes longer primary cilia both in vitro and in vivo. Strikingly, these Notch-regulated effects are Shh independent. These data identify Notch signalling as a novel modulator of Shh signalling that acts mechanistically via regulation of ciliary localisation of key components of its transduction machinery.

Non-CDK-bound p27 (p27(NCDK)) is a marker for cell stress and is regulated through the Akt/PKB and AMPK-kinase pathways.

p27Kip1 (p27) tumour suppressor protein is regulated by multiple mechanisms including its turnover, localization and complex formation with its key targets, cyclin-dependent kinases (CDK) and cyclins. We have earlier shown that p27 exists in cells in a form that lacks cyclin/CDK interactions (hence non-CDK, p27(NCDK)) but the nature of p27(NCDK) has remained unresolved. Here we demonstrate that the epitope recognized by the p27(NCDK)-specific antibody resides in the p27 CDK-interaction domain and that p27(NCDK) is regulated by the balance of CDK inhibitors and cyclin-CDK complexes. We find that signalling by cellular growth promoting pathways, like phosphoinositol 3-kinase (PI3K) and specifically Akt/PKB kinase, inversely correlates with p27(NCDK) levels whereas total p27 levels are unaffected. p27(NCDK), but not total p27, is increased by cellular perturbations such as hyperosmotic and metabolic stress and activation of AMP-activated protein kinase (AMPK). By using AMPK catalytic subunit proficient and deficient cells we further demonstrate that the AMPK pathway governs p27(NCDK) responses to metabolic stress and PI3K inhibition. These results indicate that p27(NCDK) is a sensitive marker for both cell stress and proliferation over and above p27 and is regulated by Akt/PKB and AMPK pathways.

The phosphatidylinositol 3-kinase/Akt pathway regulates transforming growth factor-{beta} signaling by destabilizing ski and inducing Smad7.

Ski is an oncoprotein that negatively regulates transforming growth factor (TGF)-beta signaling. It acts as a transcriptional co-repressor by binding to TGF-beta signaling molecules, Smads. Efficient TGF-beta signaling is facilitated by rapid proteasome-mediated degradation of Ski by TGF-beta. Here we report that Ski is phosphorylated by Akt/PKB kinase. Akt phosphorylates Ski on a highly conserved Akt motif at threonine 458 both in vitro and in vivo. The phosphorylation of Ski at threonine 458 is induced by Akt pathway activators including insulin, insulin-like growth factor-1, and hepatocyte growth factor. The phosphorylation of Ski causes its destabilization and reduces Ski-mediated inhibition of expression of another negative regulator of TGF-beta, Smad7. Induction of Smad7 levels leads to inactivation of TGF-beta receptors and TGF-beta signaling cascade, as indicated by reduced induction of TGF-beta target p15. Therefore, Akt modulates TGF-beta signaling by temporarily adjusting the levels of two TGF-beta pathway negative regulators, Ski and Smad7. These novel findings demonstrate that Akt pathway activation directly impacts TGF-beta pathway.

A balance of positive and negative regulators determines the pace of the segmentation clock.

Somitogenesis is regulated by a molecular oscillator that drives dynamic gene expression within the pre-somitic mesoderm. Previous mathematical models of the somitogenesis clock that invoke the mechanism of delayed negative feedback predict that its oscillation period depends on the sum of delays inherent to negative-feedback loops and inhibitor half-lives. We develop a mathematical model that explores the possibility that positive feedback also plays a role in determining the period of clock oscillations. The model predicts that increasing the half-life of the positive regulator, Notch intracellular domain (NICD), can lead to elevated NICD levels and an increase in the oscillation period. To test this hypothesis, we investigate a phenotype induced by various small molecule inhibitors in which the clock is slowed. We observe elevated levels and a prolonged half-life of NICD. Reducing NICD production rescues these effects. These data provide the first indication that tight control of the turnover of positive as well as negative regulators of the clock determines its periodicity.