Selective PP2A Enhancement through Biased Heterotrimer Stabilization.

Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory "B" subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.

Global phosphoproteomics of CCR5-tropic HIV-1 signaling reveals reprogramming of cellular protein production pathways and identifies p70-S6K1 and MK2 as HIV-responsive kinases required for optimal infection of CD4+ T cells.

BACKGROUND: Viral reprogramming of host cells enhances replication and is initiated by viral interaction with the cell surface. Upon human immunodeficiency virus (HIV) binding to CD4+ T cells, a signal transduction cascade is initiated that reorganizes the actin cytoskeleton, activates transcription factors, and alters mRNA splicing pathways. METHODS: We used a quantitative mass spectrometry-based phosphoproteomic approach to investigate signal transduction cascades initiated by CCR5-tropic HIV, which accounts for virtually all transmitted viruses and the vast majority of viruses worldwide. RESULTS: CCR5-HIV signaling induced significant reprogramming of the actin cytoskeleton and mRNA splicing pathways, as previously described. In addition, CCR5-HIV signaling induced profound changes to the mRNA transcription, processing, translation, and post-translational modifications pathways, indicating that virtually every stage of protein production is affected. Furthermore, we identified two kinases regulated by CCR5-HIV signaling-p70-S6K1 (RPS6KB1) and MK2 (MAPKAPK2)-that were also required for optimal HIV infection of CD4+ T cells. These kinases regulate protein translation and cytoskeletal architecture, respectively, reinforcing the importance of these pathways in viral replication. Additionally, we found that blockade of CCR5 signaling by maraviroc had relatively modest effects on CCR5-HIV signaling, in agreement with reports that signaling by CCR5 is dispensable for HIV infection but in contrast to the critical effects of CXCR4 on cortical actin reorganization. CONCLUSIONS: These results demonstrate that CCR5-tropic HIV induces significant reprogramming of host CD4+ T cell protein production pathways and identifies two novel kinases induced upon viral binding to the cell surface that are critical for HIV replication in host cells.

Phosphoproteomics Profiling of Nonsmall Cell Lung Cancer Cells Treated with a Novel Phosphatase Activator.

Activation of protein phosphatase 2A (PP2A) is a promising anticancer therapeutic strategy, as this tumor suppressor has the ability to coordinately downregulate multiple pathways involved in the regulation of cellular growth and proliferation. In order to understand the systems-level perturbations mediated by PP2A activation, we carried out mass spectrometry-based phosphoproteomic analysis of two KRAS mutated non-small cell lung cancer (NSCLC) cell lines (A549 and H358) treated with a novel small molecule activator of PP2A (SMAP). Overall, this permitted quantification of differential signaling across over 1600 phosphoproteins and 3000 phosphosites. Kinase activity assessment and pathway enrichment implicate collective downregulation of RAS and cell cycle kinases in the case of both cell lines upon PP2A activation. However, the effects on RAS-related signaling are attenuated for A549 compared to H358, while the effects on cell cycle-related kinases are noticeably more prominent in A549. Network-based analyses and validation experiments confirm these detailed differences in signaling. These studies reveal the power of phosphoproteomics studies, coupled to computational systems biology, to elucidate global patterns of phosphatase activation and understand the variations in response to PP2A activation across genetically similar NSCLC cell lines.

The KSEA App: a web-based tool for kinase activity inference from quantitative phosphoproteomics.

Summary: Computational characterization of differential kinase activity from phosphoproteomics datasets is critical for correctly inferring cellular circuitry and how signaling cascades are altered in drug treatment and/or disease. Kinase-Substrate Enrichment Analysis (KSEA) offers a powerful approach to estimating changes in a kinase's activity based on the collective phosphorylation changes of its identified substrates. However, KSEA has been limited to programmers who are able to implement the algorithms. Thus, to make it accessible to the larger scientific community, we present a web-based application of this method: the KSEA App. Overall, we expect that this tool will offer a quick and user-friendly way of generating kinase activity estimates from high-throughput phosphoproteomics datasets. Availability and Implementation: the KSEA App is a free online tool: casecpb.shinyapps.io/ksea/. The source code is on GitHub: github.com/casecpb/KSEA/. The application is also available as the R package "KSEAapp" on CRAN: CRAN.R-project.org/package=KSEAapp/. Contact: mark.chance@case.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Activation of tumor suppressor protein PP2A inhibits KRAS-driven tumor growth.

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.