Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2.

Protein phosphatase 2A (PP2A) enzymes can suppress tumors, but they are often inactivated in human cancers overexpressing inhibitory proteins. Here, we identify a class of small-molecule iHAPs (improved heterocyclic activators of PP2A) that kill leukemia cells by allosterically assembling a specific heterotrimeric PP2A holoenzyme consisting of PPP2R1A (scaffold), PPP2R5E (B56ε, regulatory), and PPP2CA (catalytic) subunits. One compound, iHAP1, activates this complex but does not inhibit dopamine receptor D2, a mediator of neurologic toxicity induced by perphenazine and related neuroleptics. The PP2A complex activated by iHAP1 dephosphorylates the MYBL2 transcription factor on Ser241, causing irreversible arrest of leukemia and other cancer cells in prometaphase. In contrast, SMAPs, a separate class of compounds, activate PP2A holoenzymes containing a different regulatory subunit, do not dephosphorylate MYBL2, and arrest tumor cells in G1 phase. Our findings demonstrate that small molecules can serve as allosteric switches to activate distinct PP2A complexes with unique substrates.

Target-Based Discovery of an Inhibitor of the Regulatory Phosphatase PPP1R15B.

Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.

Histone phosphorylation integrates the hepatic glucagon-PKA-CREB gluconeogenesis program in response to fasting.

The glucagon-PKA signal is generally believed to control hepatic gluconeogenesis via the CREB transcription factor. Here we uncovered a distinct function of this signal in directly stimulating histone phosphorylation for gluconeogenic gene regulation in mice. In the fasting state, CREB recruited activated PKA to regions near gluconeogenic genes, where PKA phosphorylated histone H3 serine 28 (H3S28ph). H3S28ph, recognized by 14-3-3ζ, promoted recruitment of RNA polymerase II and transcriptional stimulation of gluconeogenic genes. In contrast, in the fed state, more PP2A was found near gluconeogenic genes, which counteracted PKA by dephosphorylating H3S28ph and repressing transcription. Importantly, ectopic expression of phosphomimic H3S28 efficiently restored gluconeogenic gene expression when liver PKA or CREB was depleted. These results together highlight a different functional scheme in regulating gluconeogenesis by the glucagon-PKA-CREB-H3S28ph cascade, in which the hormone signal is transmitted to chromatin for rapid and efficient gluconeogenic gene activation.

IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.

Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Sensing by Dendritic Cells in Psoriasis.

Psoriasis is a chronic inflammatory disease whose etiology is multifactorial. The contributions of cellular metabolism to psoriasis are unclear. Here, we report that interleukin-17 (IL-17) downregulated Protein Phosphatase 6 (PP6) in psoriatic keratinocytes, causing phosphorylation and activation of the transcription factor C/EBP-ß and subsequent generation of arginase-1. Mice lacking Pp6 in keratinocytes were predisposed to psoriasis-like skin inflammation. Accumulation of arginase-1 in Pp6-deficient keratinocytes drove polyamine production from the urea cycle. Polyamines protected self-RNA released by psoriatic keratinocytes from degradation and facilitated the endocytosis of self-RNA by myeloid dendritic cells to promote toll-like receptor-7 (TLR7)-dependent RNA sensing and IL-6 production. An arginase inhibitor improved skin inflammation in murine and non-human primate models of psoriasis. Our findings suggest that urea cycle hyperreactivity and excessive polyamine generation in psoriatic keratinocytes promote self-RNA sensation and PP6 deregulation in keratinocytes is a pivotal event that amplifies the inflammatory circuits in psoriasis.

Protein phosphatases in the RNAPII transcription cycle: erasers, sculptors, gatekeepers, and potential drug targets.

The transcription cycle of RNA polymerase II (RNAPII) is governed at multiple points by opposing actions of cyclin-dependent kinases (CDKs) and protein phosphatases, in a process with similarities to the cell division cycle. While important roles of the kinases have been established, phosphatases have emerged more slowly as key players in transcription, and large gaps remain in understanding of their precise functions and targets. Much of the earlier work focused on the roles and regulation of sui generis and often atypical phosphatases-FCP1, Rtr1/RPAP2, and SSU72-with seemingly dedicated functions in RNAPII transcription. Decisive roles in the transcription cycle have now been uncovered for members of the major phosphoprotein phosphatase (PPP) family, including PP1, PP2A, and PP4-abundant enzymes with pleiotropic roles in cellular signaling pathways. These phosphatases appear to act principally at the transitions between transcription cycle phases, ensuring fine control of elongation and termination. Much is still unknown, however, about the division of labor among the PPP family members, and their possible regulation by or of the transcriptional kinases. CDKs active in transcription have recently drawn attention as potential therapeutic targets in cancer and other diseases, raising the prospect that the phosphatases might also present opportunities for new drug development. Here we review the current knowledge and outstanding questions about phosphatases in the context of the RNAPII transcription cycle.

Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity.

In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals and Drosophila Genetic depletion of PPP4 shortens the circadian period, whereas overexpression lengthens it. PPP4 inhibits CLOCK/BMAL1 transactivation activity by binding to BMAL1 and counteracting its phosphorylation. This leads to increased CLOCK/BMAL1 DNA occupancy and decreased transcriptional activity, which counteracts the "kamikaze" properties of CLOCK/BMAL1. Through this mechanism, PPP4 contributes to the critical delay of negative feedback by retarding PER/CRY/CK1δ-mediated inhibition of CLOCK/BMAL1.

BMAL1 dephosphorylation determines the pace of the circadian clock.

In mammals, virtually all body cells harbor cell-autonomous and self-sustained circadian oscillators that rely on delayed negative feedback loops in gene expression. Transcriptional activation and repression play a major role in keeping these clocks ticking, but numerous post-translational mechanisms-and particularly the phosphorylation of core clock components by protein kinases-are also critically involved in setting the pace of these timekeepers. In this issue of Genes & Development, Klemz and colleagues (pp. 1161-1174) now show how dephosphorylation of BMAL1 by protein phosphatase 4 (PPP4) participates in the modulation of circadian timing.

Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of Transcription.

Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.

Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

Targeted chemotherapy overcomes drug resistance in melanoma.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAFV600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRASQ61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAFV600E PDX highlighting its effectiveness in combating the advent of drug resistance.

Structural basis of ubiquitin-independent PP1 complex disassembly by p97.

The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.

PHOSPHATASE 2A dephosphorylates PHYTOCHROME INTERACTING FACTOR3 to modulate photomorphogenesis in Arabidopsis.

The phytochrome (phy) family of sensory photoreceptors modulates developmental programs in response to ambient light. Phys also control gene expression in part by directly interacting with the bHLH class of transcription factors, PHYTOCHROME-INTERACTING FACTORS (PIFs), and inducing their rapid phosphorylation and degradation. Several kinases have been shown to phosphorylate PIFs and promote their degradation. However, the phosphatases that dephosphorylate PIFs are less understood. Here, we describe four regulatory subunits of the Arabidopsis (Arabidopsis thaliana) protein PHOSPHATASE 2A (PP2A) family (B'α, B'ß, B''α and B''ß) that interact with PIF3 in yeast two-hybrid, in vitro and in vivo assays. The pp2ab''αß and b''αß/b'αß mutants displayed short hypocotyls, while the overexpression of the B subunits induced longer hypocotyls compared to the wild type under red light. The light-induced degradation of PIF3 was faster in the b''αß/b'αß quadruple mutant compared to in the wild type. Consistently, immunoprecipitated PP2A A and B subunits directly dephosphorylated PIF3-MYC in vitro. RNA-seq analyses showed that B''α and B''ß alter global gene expression in response to red light. PIFs (PIF1, PIF3, PIF4 and PIF5) are epistatic to these B subunits in regulating hypocotyl elongation under red light. Collectively, these data show an essential function of PP2A in dephosphorylating PIF3 to modulate photomorphogenesis in Arabidopsis.

A Consensus Binding Motif for the PP4 Protein Phosphatase.

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response.

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2αP) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2αP in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2αP by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

The functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange.

To Build by Destruction.

In this issue of Molecular Cell, Weith et al. (2018) demonstrate that p97, together with a SEP adaptor, can catalyze ordered subunit exchange to facilitate the biogenesis of protein phosphatase-1 (PP1) holoenzyme, establishing a novel ubiquitin-independent "segregase" function for this versatile ATPase.

Environmental factor reversibly determines cellular identity through opposing Integrators that unify epigenetic and transcriptional pathways.

Organisms must adapt to environmental stresses to ensure their survival and prosperity. Different types of stresses, including thermal, mechanical, and hypoxic stresses, can alter the cellular state that accompanies changes in gene expression but not the cellular identity determined by a chromatin state that remains stable throughout life. Some tissues, such as adipose tissue, demonstrate remarkable plasticity and adaptability in response to environmental cues, enabling reversible cellular identity changes; however, the mechanisms underlying these changes are not well understood. We hypothesized that positive and/or negative "Integrators" sense environmental cues and coordinate the epigenetic and transcriptional pathways required for changes in cellular identity. Adverse environmental factors such as pollution disrupt the coordinated control contributing to disease development. Further research based on this hypothesis will reveal how organisms adapt to fluctuating environmental conditions, such as temperature, extracellular matrix stiffness, oxygen, cytokines, and hormonal cues by changing their cellular identities.

PP2B-Dependent Cerebellar Plasticity Sets the Amplitude of the Vestibulo-ocular Reflex during Juvenile Development.

Throughout life, the cerebellum plays a central role in the coordination and optimization of movements, using cellular plasticity to adapt a range of behaviors. Whether these plasticity processes establish a fixed setpoint during development, or continuously adjust behaviors throughout life, is currently unclear. Here, by spatiotemporally manipulating the activity of protein phosphatase 2B (PP2B), an enzyme critical for cerebellar plasticity in male and female mice, we examined the consequences of disrupted plasticity on the performance and adaptation of the vestibulo-ocular reflex (VOR). We find that, in contrast to Purkinje cell (PC)-specific deletion starting early postnatally, acute pharmacological as well as adult-onset genetic deletion of PP2B affects all forms of VOR adaptation but not the level of VOR itself. Next, we show that PC-specific genetic deletion of PP2B in juvenile mice leads to a progressive loss of the protein PP2B and a concurrent change in the VOR, in addition to the loss of adaptive abilities. Finally, re-expressing PP2B in adult mice that lack PP2B expression from early development rescues VOR adaptation but does not affect the performance of the reflex. Together, our results indicate that chronic or acute, genetic, or pharmacological block of PP2B disrupts the adaptation of the VOR. In contrast, only the absence of plasticity during cerebellar development affects the setpoint of VOR, an effect that cannot be corrected after maturation of the cerebellum. These findings suggest that PP2B-dependent cerebellar plasticity is required during a specific period to achieve the correct setpoint of the VOR.

The SDS22:PP1:I3 complex: SDS22 binding to PP1 loosens the active site metal to prime metal exchange.

SDS22 and Inhibitor-3 (I3) are two ancient regulators of protein phosphatase 1 (PP1) that regulate multiple essential biological processes. Both SDS22 and I3 form stable dimeric complexes with PP1; however, and atypically for PP1 regulators, they also form a triple complex, where both proteins bind to PP1 simultaneously (SPI complex). Here we report the crystal structure of the SPI complex. While both regulators bind PP1 in conformations identical to those observed in their individual PP1 complexes, PP1 adopts the SDS22-bound conformation, which lacks its M1 metal. Unexpectedly, surface plasmon resonance (SPR) revealed that the affinity of I3 for the SDS22:PP1 complex is ∼10-fold lower than PP1 alone. We show that this change in binding affinity is solely due to the interaction of I3 with the PP1 active site, specifically PP1's M2 metal, demonstrating that SDS22 likely allows for PP1 M2 metal exchange and thus PP1 biogenesis.