Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration.

Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior.

A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency.

The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.

The ubiquitin binding region of the Smurf HECT domain facilitates polyubiquitylation and binding of ubiquitylated substrates.

Mono- and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monoubiquitylated form of RhoA to the Smurf HECT domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT domain.

Atypical function of a centrosomal module in WNT signalling drives contextual cancer cell motility.

Centrosomes control cell motility, polarity and migration that is thought to be mediated by their microtubule-organizing capacity. Here we demonstrate that WNT signalling drives a distinct form of non-directional cell motility that requires a key centrosome module, but not microtubules or centrosomes. Upon exosome mobilization of PCP-proteins, we show that DVL2 orchestrates recruitment of a CEP192-PLK4/AURKB complex to the cell cortex where PLK4/AURKB act redundantly to drive protrusive activity and cell motility. This is mediated by coordination of formin-dependent actin remodelling through displacement of cortically localized DAAM1 for DAAM2. Furthermore, abnormal expression of PLK4, AURKB and DAAM1 is associated with poor outcomes in breast and bladder cancers. Thus, a centrosomal module plays an atypical function in WNT signalling and actin nucleation that is critical for cancer cell motility and is associated with more aggressive cancers. These studies have broad implications in how contextual signalling controls distinct modes of cell migration.

Degradation of RhoA by Smurf1 ubiquitin ligase.

The Rho family of small GTPases plays a key role in the dynamic regulation of the actin cytoskeleton that underlies various important cellular functions such as shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, could specifically target RhoA but not Cdc42 or Rac1 for degradation. Smurf1 interacts with the dominant inactive form of RhoA, RhoA N19, which binds constitutively to guanine nucleotide exchange factors (GEFs) in vivo. Smurf1 also interacts directly with either nucleotide-free or GDP-bound RhoA in vitro; however, loading with GTPgammaS inhibits the interaction. RhoA is ubiquitinated by wild-type Smurf1 but not the catalytic mutant of Smurf1 (C699A) in vivo and in vitro, indicating that RhoA is a direct substrate of Smurf1. In this chapter, we summarize the systems and methods used in the analyses of Smurf1-regulated RhoA ubiquitination and degradation.

PTEN regulates cilia through Dishevelled.

Cilia are hair-like cellular protrusions important in many aspects of eukaryotic biology. For instance, motile cilia enable fluid movement over epithelial surfaces, while primary (sensory) cilia play roles in cellular signalling. The molecular events underlying cilia dynamics, and particularly their disassembly, are not well understood. Phosphatase and tensin homologue (PTEN) is an extensively studied tumour suppressor, thought to primarily act by antagonizing PI3-kinase signalling. Here we demonstrate that PTEN plays an important role in multicilia formation and cilia disassembly by controlling the phosphorylation of Dishevelled (DVL), another ciliogenesis regulator. DVL is a central component of WNT signalling that plays a role during convergent extension movements, which we show here are also regulated by PTEN. Our studies identify a novel protein substrate for PTEN that couples PTEN to regulation of cilia dynamics and WNT signalling, thus advancing our understanding of potential underlying molecular etiologies of PTEN-related pathologies.

Switch enhancers interpret TGF-ß and Hippo signaling to control cell fate in human embryonic stem cells.

A small toolkit of morphogens is used repeatedly to direct development, raising the question of how context dictates interpretation of the same cue. One example is the transforming growth factor ß (TGF-ß) pathway that in human embryonic stem cells fulfills two opposite functions: pluripotency maintenance and mesendoderm (ME) specification. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex composed of transcriptional effectors of the Hippo pathway (TAZ/YAP/TEAD), the TGF-ß pathway (SMAD2/3), and the pluripotency regulator OCT4 (TSO). TSO collaborates with NuRD repressor complexes to buffer pluripotency gene expression while suppressing ME genes. Importantly, the SMAD DNA binding partner FOXH1, a major specifier of ME, is found near TSO elements, and upon fate specification we show that TSO is disrupted with subsequent SMAD-FOXH1 induction of ME. These studies define switch-enhancer elements and provide a framework to understand how cellular context dictates interpretation of the same morphogen signal in development.

Structural basis for specificity of TGFß family receptor small molecule inhibitors.

Transforming growth factor-ß (TGFß) receptor kinase inhibitors have a great therapeutic potential. SB431542 is one of the mainly used kinase inhibitors of the TGFß/Activin pathway receptors, but needs improvement of its EC(50) (EC(50)=1 µM) to be translated to clinical use. A key feature of SB431542 is that it specifically targets receptors from the TGFß/Activin pathway but not the closely related receptors from the bone morphogenic proteins (BMP) pathway. To understand the mechanisms of this selectivity, we solved the crystal structure of the TGFß type I receptor (TßRI) kinase domain in complex with SB431542. We mutated TßRI residues coordinating SB431542 to their counterparts in activin-receptor like kinase 2 (ALK2), a BMP receptor kinase, and tested the kinase activity of mutated TßRI. We discovered that a Ser280Thr mutation yielded a TßRI variant that was resistant to SB431542 inhibition. Furthermore, the corresponding Thr283Ser mutation in ALK2 yielded a BMP receptor sensitive to SB431542. This demonstrated that Ser280 is the key determinant of selectivity for SB431542. This work provides a framework for optimising the SB431542 scaffold to more potent and selective inhibitors of the TGFß/Activin pathway.

Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain.

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.

Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain.

The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFbeta signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT domain class, to the TGFbeta receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT domain that functions independently of Smad7 and potently inhibits TGFbeta signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.

Heterologous expression of cry2 gene from a local strain of Bacillus thuringiensis isolated in Nigeria.

A cry2 gene encoding a larvicidal crystal protein was isolated from a strain of Bacillus thuringiensis found in soil samples in Nigeria. This gene was cloned into plasmid pUC19 and subcloned into both pBluescript (sk(+/-)) and pPICZ alpha B placed under a T7/AOXI (alcohol oxidase I) promoter respectively and transformed into Escherichia coli and Pichia pastoris. Clones were induced for expression, and the cellular proteins extracted and analysed by SDS/PAGE. Integration of an insert into the yeast chromosome was confirmed by PCR amplification using AOXI primers designed to monitor the intactness of the insertion into the chromosome. The expression cassettes constructed were both expressed in E. coli strain (XL1-blue) and P. pastoris (SMD1168) respectively. An approximately 70 kDa recombinant toxin was obtained both in P. pastoris and E. coli in different quantities. Expression was confirmed by Northern-blot analysis of 2.0 kb transcripts, obtained from clones induced for RNA transcripts, which hybridized with a [(32)P]dCTP-labelled probe prepared from a 641 bp fragment of restriction-endonuclease- Hae II-digested PCR product of the cry2 gene.

Regulation of cell polarity and protrusion formation by targeting RhoA for degradation.

The Rho family of small guanosine triphosphatases regulates actin cytoskeleton dynamics that underlie cellular functions such as cell shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, regulated cell polarity and protrusive activity and was required to maintain the transformed morphology and motility of a tumor cell. Atypical protein kinase C zeta (PKCzeta), an effector of the Cdc42/Rac1-PAR6 polarity complex, recruited Smurf1 to cellular protrusions, where it controlled the local level of RhoA. Smurf1 thus links the polarity complex to degradation of RhoA in lamellipodia and filopodia to prevent RhoA signaling during dynamic membrane movements.