Small molecule activators of pre-mRNA 3' cleavage.

3' Cleavage and polyadenylation are obligatory steps in the biogenesis of most mammalian pre-mRNAs. In vitro reconstitution of the 3' cleavage reaction from human cleavage factors requires high concentrations of creatine phosphate (CP), though how CP activates cleavage is not known. Previously, we proposed that CP might work by competitively inhibiting a cleavage-suppressing serine/threonine (S/T) phosphatase. Here we show that fluoride/EDTA, a general S/T phosphatase inhibitor, activates in vitro cleavage in place of CP. Subsequent testing of inhibitors specific for different S/T phosphatases showed that inhibitors of the PPM family of S/T phosphatases, which includes PP2C, but not the PPP family, which includes PP1, PP2A, and PP2B, activated 3' cleavage in vitro. In particular, NCI 83633, an inhibitor of PP2C, activated extensive 3' cleavage at a concentration 50-fold below that required by fluoride or CP. The testing of structural analogs led to the identification of a more potent compound that activated 3' cleavage at 200 microM. While testing CP analogs to understand the origin of its cleavage activation effect, we found phosphocholine to be a more effective activator than CP. The minimal structural determinants of 3' cleavage activation by phosphocholine were identified. Our results describe a much improved small molecule activator of in vitro pre-mRNA cleavage, identify the molecular determinants of cleavage activation by phosphoamines such as phosphocholine, and suggest that a PPM family phosphatase is involved in the negative regulation of mammalian pre-mRNA 3' cleavage.

The Rho-specific GEF Lfc interacts with neurabin and spinophilin to regulate dendritic spine morphology.

Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.

Independent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiproliferative effects of indirubins.

Indirubin, a bis-indole obtained from various natural sources, is responsible for the reported antileukemia activity of a Chinese Medicinal recipe, Danggui Longhui Wan. However, its molecular mechanism of action is still not well understood. In addition to inhibition of cyclin-dependent kinases and glycogen synthase kinase-3, indirubins have been reported to activate the aryl hydrocarbon receptor (AhR), a cotranscriptional factor. Here, we confirm the interaction of AhR and indirubin using a series of indirubin derivatives and show that their binding modes to AhR and to protein kinases are unrelated. As reported for other AhR ligands, binding of indirubins to AhR leads to its nuclear translocation. Furthermore, the apparent survival of AhR-/- and +/+ cells, as measured by the MTT assay, is equally sensitive to the kinase-inhibiting indirubins. Thus, the cytotoxic effects of indirubins are AhR-independent and more likely to be linked to protein kinase inhibition. In contrast, a dramatic cytostatic effect, as measured by actual cell counts and associated with a sharp G1 phase arrest, is induced by 1-methyl-indirubins, a subfamily of AhR-active but kinase-inactive indirubins. As shown for TCDD (dioxin), this effect appears to be mediated through the AhR-dependent expression of p27(KIP1). Altogether these results suggest that AhR activation, rather than kinase inhibition, is responsible for the cytostatic effects of some indirubins. In contrast, kinase inhibition, rather than AhR activation, represents the main mechanism underlying the cytotoxic properties of this class of promising antitumor molecules.

GSK-3-selective inhibitors derived from Tyrian purple indirubins.

Gastropod mollusks have been used for over 2500 years to produce the "Tyrian purple" dye made famous by the Phoenicians. This dye is constituted of mixed bromine-substituted indigo and indirubin isomers. Among these, the new natural product 6-bromoindirubin and its synthetic, cell-permeable derivative, 6-bromoindirubin-3'-oxime (BIO), display remarkable selective inhibition of glycogen synthase kinase-3 (GSK-3). Cocrystal structure of GSK-3beta/BIO and CDK5/p25/indirubin-3'-oxime were resolved, providing a detailed view of indirubins' interactions within the ATP binding pocket of these kinases. BIO but not 1-methyl-BIO, its kinase inactive analog, also inhibited the phosphorylation on Tyr276/216, a GSK-3alpha/beta activation site. BIO but not 1-methyl-BIO reduced beta-catenin phosphorylation on a GSK-3-specific site in cellular models. BIO but not 1-methyl-BIO closely mimicked Wnt signaling in Xenopus embryos. 6-bromoindirubins thus provide a new scaffold for the development of selective and potent pharmacological inhibitors of GSK-3.