Nathan D. Camp

Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling

Aberrant WNT signal transduction is involved in many diseases. In colorectal cancer and melanoma, mutational disruption of proteins involved in the degradation of β-catenin, the key effector of the WNT signaling pathway, results in stabilization of β-catenin and, in turn, activation of transcription. We have used tandem-affinity protein purification and mass spectrometry to define the protein interaction network of the β-catenin destruction complex. This assay revealed that WTX, a protein encoded by a gene mutated in Wilms tumors, forms a complex with β-catenin, AXIN1, β-TrCP2 (β-transducin repeat–containing protein 2), and APC (adenomatous polyposis coli). Functional analyses in cultured cells, Xenopus, and zebrafish demonstrate that WTX promotes β-catenin ubiquitination and degradation, which antagonize WNT/β-catenin signaling. These data provide a possible mechanistic explanation for the tumor suppressor activity of WTX.

Small-molecule synergist of the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway regulates cell fate and behavior during embryogenesis, adult tissue homeostasis, and regeneration. When inappropriately activated, the pathway has been linked to colorectal cancer and melanoma, and when attenuated it may contribute to Alzheimers disease and osteoporosis. Small molecules that modulate Wnt signaling will likely provide new insights into the regulation of this key developmental pathway and ultimately provide pharmacological agents to control Wnt signaling in vivo. To this end, we screened a library of 100,000 small molecules for activity in a cell-based assay of Wnt/β-catenin signaling and discovered a purine derivative, QS11, that synergizes with Wnt-3a ligand in the activation of Wnt/β-catenin signal transduction. Through affinity chromatography and subsequent functional assays, we showed that QS11 binds and inhibits the GTPase activating protein of ADP-ribosylation factor 1 (ARFGAP1), suggesting that QS11 modulates Wnt/β-catenin signaling through an effect on protein trafficking. Consistent with its function as an ARFGAP inhibitor, QS11 inhibits migration of ARFGAP overexpressing breast cancer cells.

Wilms Tumor Gene on X Chromosome (WTX) Inhibits Degradation of NRF2 Protein through Competitive Binding to KEAP1 Protein

Background: KEAP1 is a ubiquitin ligase adaptor that promotes the ubiquitination and degradation of NRF2, a transcription factor that drives the antioxidant response. Results: Wilms tumor gene on the X chromosome (WTX) stabilizes NRF2 by competing with NRF2 for binding to KEAP1. Conclusion: WTX regulates the antioxidant response. Significance: This study reveals a novel regulatory mechanism governing the antioxidant response. WTX is a tumor suppressor protein that is lost or mutated in up to 30% of cases of Wilms tumor. Among its known functions, WTX interacts with the β-transducin repeat containing family of ubiquitin ligase adaptors and promotes the ubiquitination and degradation of the transcription factor β-catenin, a key control point in the WNT/β-catenin signaling pathway. Here, we report that WTX interacts with a second ubiquitin ligase adaptor, KEAP1, which functions to regulate the ubiquitination of the transcription factor NRF2, a key control point in the antioxidant response. Surprisingly, we find that unlike its ability to promote the ubiquitination of β-catenin, WTX inhibits the ubiquitination of NRF2. WTX and NRF2 compete for binding to KEAP1, and thus loss of WTX leads to rapid ubiquitination and degradation of NRF2 and a reduced response to cytotoxic insult. These results expand our understanding of the molecular mechanisms of WTX and reveal a novel regulatory mechanism governing the antioxidant response.

Bruton's tyrosine kinase revealed as a negative regulator of wnt-b-catenin signaling

Combining an siRNA screen with a small-molecule screen reveals BTK as a nuclear inhibitor of the Wnt–β-catenin pathway. BTK Checks Wnt–β-Catenin–Mediated Gene Expression Dysregulated Wnt signaling is associated with several human diseases. James et al. now connect the Wnt–β-catenin pathway to Bruton’s tyrosine kinase, which is encoded by the gene responsible for X-linked agammaglobulinemia, a disease associated with decreased ability to fight infection due to a deficiency in B cells. By combining a small-molecule screen with a targeted siRNA screen, BTK was identified as an inhibitor of β-catenin–mediated gene expression. BTK did not alter the abundance of β-catenin in the presence or absence of Wnt; instead, it appeared to influence the stability of CDC73, a constituent of the PAF elongation complex and known binding partner of β-catenin. In B cells, CDC73 also inhibited β-catenin–mediated gene expression and BTK may act through this nuclear protein to restrain β-catenin’s transcriptional activity. Wnts are secreted ligands that activate several receptor-mediated signal transduction cascades. Homeostatic Wnt signaling through β-catenin is required in adults, because either elevation or attenuation of β-catenin function has been linked to diverse diseases. To contribute to the identification of both protein and pharmacological regulators of this pathway, we describe a combinatorial screen that merged data from a high-throughput screen of known bioactive compounds with an independent focused small interfering RNA screen. Each screen independently revealed Bruton’s tyrosine kinase (BTK) as an inhibitor of Wnt–β-catenin signaling. Loss of BTK function in human colorectal cancer cells, human B cells, zebrafish embryos, and cells derived from X-linked agammaglobulinemia patients with a mutant BTK gene resulted in elevated Wnt–β-catenin signaling, confirming that BTK acts as a negative regulator of this pathway. From affinity purification–mass spectrometry and biochemical binding studies, we found that BTK directly interacts with a nuclear component of Wnt–β-catenin signaling, CDC73. Further, we show that BTK increased the abundance of CDC73 in the absence of stimulation and that CDC73 acted as a repressor of β-catenin–mediated transcription in human colorectal cancer cells and B cells.

WIKI4, a Novel Inhibitor of Tankyrase and Wnt/ß-Catenin Signaling

The Wnt/ß-catenin signaling pathway controls important cellular events during development and often contributes to disease when dysregulated. Using high throughput screening we have identified a new small molecule inhibitor of Wnt/ß-catenin signaling, WIKI4. WIKI4 inhibits expression of ß-catenin target genes and cellular responses to Wnt/ß-catenin signaling in cancer cell lines as well as in human embryonic stem cells. Furthermore, we demonstrate that WIKI4 mediates its effects on Wnt/ß-catenin signaling by inhibiting the enzymatic activity of TNKS2, a regulator of AXIN ubiquitylation and degradation. While TNKS has previously been shown to be the target of small molecule inhibitors of Wnt/ß-catenin signaling, WIKI4 is structurally distinct from previously identified TNKS inhibitors.

Integrated systems biology analysis of KSHV latent infection reveals viral induction and reliance on peroxisome mediated lipid metabolism

Kaposi’s Sarcoma associated Herpesvirus (KSHV), an oncogenic, human gamma-herpesvirus, is the etiological agent of Kaposi’s Sarcoma the most common tumor of AIDS patients world-wide. KSHV is predominantly latent in the main KS tumor cell, the spindle cell, a cell of endothelial origin. KSHV modulates numerous host cell-signaling pathways to activate endothelial cells including major metabolic pathways involved in lipid metabolism. To identify the underlying cellular mechanisms of KSHV alteration of host signaling and endothelial cell activation, we identified changes in the host proteome, phosphoproteome and transcriptome landscape following KSHV infection of endothelial cells. A Steiner forest algorithm was used to integrate the global data sets and, together with transcriptome based predicted transcription factor activity, cellular networks altered by latent KSHV were predicted. Several interesting pathways were identified, including peroxisome biogenesis. To validate the predictions, we showed that KSHV latent infection increases the number of peroxisomes per cell. Additionally, proteins involved in peroxisomal lipid metabolism of very long chain fatty acids, including ABCD3 and ACOX1, are required for the survival of latently infected cells. In summary, novel cellular pathways altered during herpesvirus latency that could not be predicted by a single systems biology platform, were identified by integrated proteomics and transcriptomics data analysis and when correlated with our metabolomics data revealed that peroxisome lipid metabolism is essential for KSHV latent infection of endothelial cells.

Endogenous N-terminal domain cleavage modulates α1D-adrenergic receptor pharmacodynamics

The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu90/Val91 in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1–91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1–91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties.

Individual protomers of a G protein-coupled receptor dimer integrate distinct functional modules

Recent advances in proteomic technology reveal G-protein-coupled receptors (GPCRs) are organized as large, macromolecular protein complexes in cell membranes, adding a new layer of intricacy to GPCR signaling. We previously reported the α1D-adrenergic receptor (ADRA1D)—a key regulator of cardiovascular, urinary and CNS function—binds the syntrophin family of PDZ domain proteins (SNTA, SNTB1, and SNTB2) through a C-terminal PDZ ligand interaction, ensuring receptor plasma membrane localization and G-protein coupling. To assess the uniqueness of this novel GPCR complex, 23 human GPCRs containing Type I PDZ ligands were subjected to TAP/MS proteomic analysis. Syntrophins did not interact with any other GPCRs. Unexpectedly, a second PDZ domain protein, scribble (SCRIB), was detected in ADRA1D complexes. Biochemical, proteomic, and dynamic mass redistribution analyses indicate syntrophins and SCRIB compete for the PDZ ligand, simultaneously exist within an ADRA1D multimer, and impart divergent pharmacological properties to the complex. Our results reveal an unprecedented modular dimeric architecture for the ADRA1D in the cell membrane, providing unexpected opportunities for fine-tuning receptor function through novel protein interactions in vivo, and for intervening in signal transduction with small molecules that can stabilize or disrupt unique GPCR:PDZ protein interfaces.

A computationally engineered RAS rheostat reveals RAS–ERK signaling dynamics

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here, we report a computationally-guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop Chemically Inducible Activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS/ERK signaling dynamics compared to growth factor stimulation, and that these dynamics differ between cell types. We also found that the clinically-approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach to design intramolecularly-regulated protein tools by applying this methodology to the Rho Family GEFs.

Dynamic mass redistribution reveals diverging importance of PDZ-ligands for G protein-coupled receptor pharmacodynamics

G protein-coupled receptors (GPCRs) are essential membrane proteins that facilitate cell-to-cell communication and co-ordinate physiological processes. At least 30 human GPCRs contain a Type I PSD-95/DLG/Zo-1 (PDZ) ligand in their distal C-terminal domain; this four amino acid motif of X-[S/T]-X-[φ] sequence facilitates interactions with PDZ domain-containing proteins. Because PDZ protein interactions have profound effects on GPCR ligand pharmacology, cellular localization, signal-transduction effector coupling and duration of activity, we analyzed the importance of Type I PDZ ligands for the function of 23 full-length and PDZ-ligand truncated (ΔPDZ) human GPCRs in cultured human cells. SNAP-epitope tag polyacrylamide gel electrophoresis revealed most Type I PDZ GPCRs exist as both monomers and multimers; removal of the PDZ ligand played minimal role in multimer formation. Additionally, SNAP-cell surface staining indicated removal of the PDZ ligand had minimal effects on plasma membrane localization for most GPCRs examined. Label-free dynamic mass redistribution functional responses, however, revealed diverging effects of the PDZ ligand. While no clear trend was observed across all GPCRs tested or even within receptor families, a subset of GPCRs displayed diminished agonist efficacy in the absence of a PDZ ligand (i.e. HT2RB, ADRB1), whereas others demonstrated enhanced agonist efficacies (i.e. LPAR2, SSTR5). These results demonstrate the utility of label-free functional assays to tease apart the contributions of conserved protein interaction domains for GPCR signal-transduction coupling in cultured cells.