The identification of selective small-molecule inhibitors can greatly aid the investigation of intracellular signaling pathways. Here, we continue our mode of action studies of TNP-470 by combining model organism chemical genetic and traditional genetics approaches to demonstrate that the target of TNP-470, MetAP-2, is essential for noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis. Given the several shared components between noncanonical and canonical Wnt/β-catenin signaling pathways, our finding that TNP-470 selectively inhibits noncanonical Wnt signaling over canonical Wnt signaling in a MetAP-2-dependent manner will aid the investigation of the diverse biological systems mediated by these important signaling pathways.
Our studies narrow the point at which MetAP-2 function is required in this signaling cascade. First, we demonstrated that MetAP-2 is downstream of Wnt and Fz, since noncanonical Wnt signaling initiated by the isoproterenol-inducible chimeric Fz2 receptor was inhibited by loss of MetAP-2 activity. While downstream of Fz, our data show that MetAP-2 is upstream of JNK and CaMKII activation. We then investigated signaling more proximal to Fz, and we showed that ΔDIX-Dvl was able to convey partial resistance to TNP-470-mediated cell cycle arrest, suggesting that MetAP-2 function is required upstream or at the level of Dishevelled. Our finding that TNP-470 inhibits noncanonical Wnt signaling between Frizzled receptors and Dishevelled further highlights the fact that this is a critical step at which canonical and noncanonical Wnt pathways diverge and is consistent with genetic data from Drosophila
A critical question left open concerns the substrate for MetAP-2 that is essential for noncanonical signaling. Previous studies have implicated members of the pertussis-sensitive Gi/o
family of heterotrimeric G proteins downstream of Fz receptors [24
]. In addition, Fz-mediated calcium release was shown to be pertussis sensitive [25
]. Interestingly, Go
, which is expressed in endothelial cells [39
], is myristoylated and requires the action of a methionine aminopeptidase to expose the penultimate glycine residue for myristic acid coupling to its amino terminus. It is tempting to speculate that retention of the Go
amino-terminal methionine upon TNP-470-mediated MetAP-2 inhibition could lead to a dominant-negative Go
protein that subsequently blocks noncanonical Wnt signaling. Experiments to test this hypothesis are underway.
Another possible connection between MetAP-2 activity and noncanonical Wnt signaling may be via regulation of protein phosphatase PP2A. Previous studies have implicated a PP2A inhibitor as a substrate for methionine aminopeptidase activity [40
]. Using a nonspecific MetAP inhibitor, bengamide E (3), Towbin and colleagues identified nine proteins whose mobility was altered upon MetAP inhibition. One of these proteins, a 42 kDa PP2A inhibitor, becomes more basic upon bengamide E incubation, consistent with the gain of a charged methionine amino terminus. Interestingly, widerborst
), a Drosophila
mutant with aberrant planar polarization in wing epithelial cells was identified as a B′ regulatory subunit of PP2A [41
]. This PP2A regulatory protein asymmetrically localizes to the distal side of a planar web of microtubules in the Drosophila
wing epithelium. Genetic studies have shown wdb
to be a conserved component of planar cell polarity in both Drosophila
and zebrafish. It is interesting to note that, like Frizzled and Dishevelled, PP2A is another shared component in both canonical and noncanonical Wnt pathways. Studies have shown that PP2A is required for Wnt/β-catenin signaling in the developing embryo [42
]. While only speculative at this moment, it is possible that retention of the amino-terminal methionine of the PP2A inhibitor upon MetAP-2 inhibition could result in aberrant PP2A regulation, leading to loss of noncanonical Wnt signaling without affecting Wnt/β-catenin signaling. Epistasis studies with Drosophila MetAP-2
mutants may provide a connection between MetAP-2
. It is interesting to note that other genetic evidence, consistent with our conclusion that MetAP-2 plays an important role in noncanonical Wnt signaling, already exists. In support of our zebrafish and cell culture data, previous studies have shown that loss of MetAP-2
leads to an embryonic lethal phenotype; however, a weaker allele results in loss of ommatidial dorsal/ventral patterning in the eye, a characteristic phenotype of noncanonical Wnt signaling misregulation [44
While other small-molecule agonists and antagonists of Wnt signaling have been reported [17
], we show that TNP-470 is the first, to our knowledge, small molecule capable of regulating noncanonical, but not canonical, Wnt signaling. Our finding that this potent antiangiogenic compound blocks noncanonical Wnt signaling suggests that this pathway may play a role in angiogenesis, which would provide many new potential drug targets for antiangiogenic therapeutic development.