Rodolfo Marquez

Role that phosphorylation of GSK3 plays in insulin and Wnt signalling defined by knockin analysis

The inactivation of glycogen synthase kinase (GSK)3 has been proposed to play important roles in insulin and Wnt signalling. To define the role that inactivation of GSK3 plays, we generated homozygous knockin mice in which the protein kinase B phosphorylation sites on GSK3α (Ser21) and GSK3β (Ser9) were changed to Ala. The knockin mice were viable and were not diabetic. Using these mice we show that inactivation of GSK3β rather than GSK3α is the major route by which insulin activates muscle glycogen synthase. In contrast, we demonstrate that the activation of muscle glycogen synthase by contraction, the stimulation of muscle glucose uptake by insulin, or the activation of hepatic glycogen synthase by glucose do not require GSK3 phosphorylation on Ser21/Ser9. GSK3 also becomes inhibited in the Wnt‐signalling pathway, by a poorly defined mechanism. In GSK3α/GSK3β homozygous knockin cells, Wnt3a induces normal inactivation of GSK3, as judged by the stabilisation of β‐catenin and stimulation of Wnt‐dependent transcription. These results establish the function of Ser21/Ser9 phosphorylation in several processes in which GSK3 inactivation has previously been implicated.

VX-680 Inhibits Aurora A and Aurora B Kinase Activity in Human Cells

VX-680, also known as MK-0457, is a member of a diverse group of small molecules that inhibit the Aurora kinases, and has shown significant potential as an anti-cancer agent. In keeping with many protein kinase inhibitors, this compound is not a monospecific agent, and its cellular specificity remains largely unknown. In cells, VX-680 blocks mitotic Histone H3 phosphorylation and induces polyploidy and apoptosis, consistent with inhibition of the mitotic protein kinase Aurora B. In this study, we have investigated the effects of VX-680 in proliferating human cancer cells, and demonstrate that it blocks the phosphorylation and activation of both Aurora A and B. Additionally, VX-680 suppresses the phosphorylation of specific substrates of each enzyme, including the Aurora A target TACC3 on Ser558. Exposure to VX-680 induces a monopolar spindle phenotype, delays mitotic progression and rapidly overrides the spindle assembly checkpoint in the presence of spindle poisons. VX-680 also exhibits potent cytotoxicity when compared to the well documented Aurora B inhibitor ZM447439. Taken together, these data identify Aurora A and Aurora B as dual intracellular targets of VX-680.

Recent advances in biomimetic natural product synthesis

This review highlights some of the most elegant and instructive biomimetic syntheses of natural products over the last few years, providing an updated overview of this area of research.

Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

BackgroundHepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor binding protein-1 (IGFBP-1), is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the T hymine-rich I nsulin R esponse E lement (TIRE). The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase). However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3) is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase), whose products are required for gluconeogenesis.ResultsIn this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression.ConclusionsThese results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

Rearrangement of epoxides in non-aldol aldol process: Allylic vs. Tertiary and secondary carbocationic centers

Abstract It has been shown that allylic carbocations are formed in preference to tertiary or secondary carbocations in the rearrangement of substituted epoxides. However protection of the alkene as a bromo ether allows for the desired rearrangement and production of intermediates for the synthesis of the cytotoxic tedanolides.

Regiocontrolled rearrangement of isobenzofurans

The regioselective alkylation and oxidative rearrangement of isobenzofurans has been achieved to generate substituted 4,8-dihydroxyisochromanones in good yields and with complete regiocontrol.

Oxidative rearrangements of isobenzofurans: Studies toward the synthesis of the ajudazols

We present a new facet of isobenzofuran chemistry which allows for its efficient manipulation to generate biologically relevant entities. This methodology has been successfully applied toward the synthesis of ajudazol A.

BCR kinase phosphorylates 14‐3‐3 Tau on residue 233

The breakpoint cluster region protein, BCR, has protein kinase activity that can auto‐ and trans‐phosphorylate serine, threonine and tyrosine residues. BCR has been implicated in chronic myelogenous leukaemia as well as important signalling pathways, and as such its interaction with 14‐3‐3 is of major interest. 14‐3‐3τ and ζ isoforms have been shown previously to be phosphorylated in vitro and in vivo by BCR kinase on serine and threonine residue(s) but site(s) were not determined. Phosphorylation of 14‐3‐3 isoforms at distinct sites is an important mode of regulation that negatively affects interaction with Raf kinase and Bax, and potentially influences the dimerization of 14‐3‐3. In this study we have further characterized the BCR−14‐3‐3 interaction and have identified the site phosphorylated by BCR. We show here that BCR interacts with at least five isoforms of 14‐3‐3 in vivo and phosphorylates 14‐3‐3τ on Ser233 and to a lesser extent 14‐3‐3ζ on Thr233. We have previously shown that these two isoforms are also phosphorylated at this site by casein kinase 1, which, in contrast to BCR, preferentially phosphorylates 14‐3‐3ζ.

Studies towards the biomimetic synthesis of bisesquiterpene lactones

A possible biomimetic connection between atractylolide, hydroxyatractylolide and biatractylolide and biepiasterolide has been demonstrated by efficiently generating the biatractylolide and biepiasterolide core structures from atractylolide and hydroxyatractylolide model butenolides via a radicaloid intermediate in a single step in good yield.

Fast and efficient synthesis of novel fumagillin analogues

Abstract A novel class of non-carbocyclic ring analogues of the potent angiogenesis inhibitors fumagillin and TNP-470 have been enantioselectively and efficiently synthesised starting from pyridine-2-thiol in 10 steps in excellent overall yield.