Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance.

Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 residue of the C subunit, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). Decreased methyl-PP2A-C levels in prostate tumors is associated with biochemical recurrence and metastasis. Silencing LCMT1 increases AR activity and promotes castration-resistant prostate cancer growth. LCMT1-dependent methyl-sensitive AB56αCme heterotrimers target AR and its critical coactivator MED1 for dephosphorylation, resulting in the eviction of the AR-MED1 complex from chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by S6K1-mediated phosphorylation-induced degradation requiring the ß-TRCP, leading to acquired resistance to anti-androgens. Finally, feedforward stabilization of LCMT1 by small molecule activator of phosphatase (SMAP) results in attenuation of AR-signaling and tumor growth inhibition in anti-androgen refractory PCa. These findings highlight methyl-PP2A-C as a prognostic marker and that the loss of LCMT1 is a major determinant in AR-addicted PCa, suggesting therapeutic potential for AR degraders or PP2A modulators in prostate cancer treatment.

The expression, localisation and interactome of pigeon CRY2.

Cryptochromes (CRY) are highly conserved signalling molecules that regulate circadian rhythms and are candidate radical pair based magnetoreceptors. Birds have at least four cryptochromes (CRY1a, CRY1b, CRY2, and CRY4), but few studies have interrogated their function. Here we investigate the expression, localisation and interactome of clCRY2 in the pigeon retina. We report that clCRY2 has two distinct transcript variants, clCRY2a, and a previously unreported splice isoform, clCRY2b which is larger in size. We show that clCRY2a mRNA is expressed in all retinal layers and clCRY2b is enriched in the inner and outer nuclear layer. To define the localisation and interaction network of clCRY2 we generated and validated a monoclonal antibody that detects both clCRY2 isoforms. Immunohistochemical studies revealed that clCRY2a/b is present in all retinal layers and is enriched in the outer limiting membrane and outer plexiform layer. Proteomic analysis showed clCRY2a/b interacts with typical circadian molecules (PER2, CLOCK, ARTNL), cell junction proteins (CTNNA1, CTNNA2) and components associated with the microtubule motor dynein (DYNC1LI2, DCTN1, DCTN2, DCTN3) within the retina. Collectively these data show that clCRY2 is a component of the avian circadian clock and unexpectedly associates with the microtubule cytoskeleton.

A phosphatase-centric mechanism drives stress signaling response.

Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomyces cerevisiae as a model system, we find that a phosphatase-driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho-proteomic response is the Endosulfine-mediated inhibition of protein phosphatase 2A-Cdc55 (PP2ACdc55 ), while we do not observe concurrent kinase activation. In fact, many of the stress-induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2ACdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well-established kinase-centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re-evaluating the impact of phosphatases on shaping the phosphorylome.

Mutation-oriented profiling of autoinhibitory kinase conformations predicts RAF inhibitor efficacies.

Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non-small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein-protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.

The biophysical, molecular, and anatomical landscape of pigeon CRY4: A candidate light-based quantal magnetosensor.

The biophysical and molecular mechanisms that enable animals to detect magnetic fields are unknown. It has been proposed that birds have a light-dependent magnetic compass that relies on the formation of radical pairs within cryptochrome molecules. Using spectroscopic methods, we show that pigeon cryptochrome clCRY4 is photoreduced efficiently and forms long-lived spin-correlated radical pairs via a tetrad of tryptophan residues. We report that clCRY4 is broadly and stably expressed within the retina but enriched at synapses in the outer plexiform layer in a repetitive manner. A proteomic survey for retinal-specific clCRY4 interactors identified molecules that are involved in receptor signaling, including glutamate receptor-interacting protein 2, which colocalizes with clCRY4. Our data support a model whereby clCRY4 acts as an ultraviolet-blue photoreceptor and/or a light-dependent magnetosensor by modulating glutamatergic synapses between horizontal cells and cones.

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.

PP2AC Phospho-Tyr307 Antibodies Are Not Specific for this Modification but Are Sensitive to Other PP2AC Modifications Including Leu309 Methylation.

Protein phosphatase 2A (PP2A) is an important regulator of signal transduction pathways and a tumor suppressor. Phosphorylation of the PP2A catalytic subunit (PP2AC) at tyrosine 307 has been claimed to inactivate PP2A and was examined in more than 180 studies using commercial antibodies, but this modification was never identified using mass spectrometry. Here we show that the most cited pTyr307 monoclonal antibodies, E155 and F-8, are not specific for phosphorylated Tyr307 but instead are hampered by PP2AC methylation at leucine 309 or phosphorylation at threonine 304. Other pTyr307 antibodies are sensitive to PP2AC methylation as well, and some cross-react with pTyr residues in general, including phosphorylated hemagglutinin tags. We identify pTyr307 using targeted mass spectrometry after transient overexpression of PP2AC and Src kinase. Yet under such conditions, none of the tested antibodies show exclusive pTyr307 specificity. Thus, data generated using these antibodies need to be revisited, and the mechanism of PP2A inactivation needs to be redefined.

Antibodies recognizing the C terminus of PP2A catalytic subunit are unsuitable for evaluating PP2A activity and holoenzyme composition.

The methyl-esterification of the C-terminal leucine of the protein phosphatase 2A (PP2A) catalytic (C) subunit is essential for the assembly of specific trimeric PP2A holoenzymes, and this region of the C subunit also contains two threonine and tyrosine phosphorylation sites. Most commercial antibodies-including the monoclonal antibody 1D6 that is part of a frequently used, commercial phosphatase assay kit-are directed toward the C terminus of the C subunit, raising questions as to their ability to recognize methylated and phosphorylated forms of the enzyme. Here, we tested several PP2A C antibodies, including monoclonal antibodies 1D6, 7A6, G-4, and 52F8 and the polyclonal antibody 2038 for their ability to specifically detect PP2A in its various modified forms, as well as to coprecipitate regulatory subunits. The tested antibodies preferentially recognized the nonmethylated form of the enzyme, and they did not coimmunoprecipitate trimeric holoenzymes containing the regulatory subunits B or B', an issue that precludes their use to monitor PP2A holoenzyme activity. Furthermore, some of the antibodies also recognized the phosphatase PP4, demonstrating a lack of specificity for PP2A. Together, these findings suggest that reinterpretation of the data generated by using these reagents is required.

The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context.

Epitope tags are short, linear antibody recognition sequences that enable detection of tagged fusion proteins by antibodies. Epitope tag position and neighboring sequences potentially affect its recognition by antibodies, and such context-dependent differences in tag binding may have a wide-ranging effect on data interpretation. We tested by Western blotting six antibodies that recognize the c-Myc epitope tag, including monoclonal antibodies 9E10, 4A6, 9B11, and 71D10 and polyclonal antibodies 9106 and A-14. All displayed context-dependent differences in their ability to detect N- or C-terminal Myc-tagged proteins. In particular, clone 9E10, the most cited Myc-tag antibody, displayed high context-dependent detection variability, whereas others, notably 4A6 and 9B11, showed much less context sensitivity in their detection of Myc-tagged proteins. The very high context sensitivity of 9E10 was further substantiated by peptide microarray analyses. We conclude that recently developed, purpose-made monoclonal antibodies specific for Myc have much more uniform reactivity in diverse assays and are much less context sensitive than is the legacy antibody 9E10.

A high sensitivity ZENK monoclonal antibody to map neuronal activity in Aves.

The transcription factor ZENK is an immediate early gene that has been employed as a surrogate marker to map neuronal activity in the brain. It has been used in a wide variety of species, however, commercially available antibodies have limited immunoreactivity in birds. To address this issue we generated a new mouse monoclonal antibody, 7B7-A3, raised against ZENK from the rock pigeon (Columba livia). We show that 7B7-A3 labels clZENK in both immunoblots and histological stainings with high sensitivity and selectivity for its target. Using a sound stimulation paradigm we demonstrate that 7B7-A3 can detect activity-dependent ZENK expression at key stations of the central auditory pathway of the pigeon. Finally, we compare staining efficiency across three avian species and confirm that 7B7-A3 is compatible with immunohistochemical detection of ZENK in the rock pigeon, zebra finch, and domestic chicken. Taken together, 7B7-A3 represents a useful tool for the avian neuroscience community to map functional activity in the brain.

Ca2+-dependent demethylation of phosphatase PP2Ac promotes glucose deprivation-induced cell death independently of inhibiting glycolysis.

Cancer cells increase glucose metabolism to support aerobic glycolysis. However, only some cancer cells are acutely sensitive to glucose withdrawal, and the underlying mechanism of this selective sensitivity is unclear. We showed that glucose deprivation initiates a cell death pathway in cancer cells that is dependent on the kinase RIPK1. Glucose withdrawal triggered rapid plasma membrane depolarization and an influx of extracellular calcium into the cell through the L-type calcium channel Cav1.3 (CACNA1D), followed by activation of the kinase CAMK1. CAMK1 and the demethylase PPME1 were required for the subsequent demethylation and inactivation of the catalytic subunit of the phosphatase PP2A (PP2Ac) and the phosphorylation of RIPK1. Plasma membrane depolarization, PP2Ac demethylation, and cell death were prevented by glucose and, unexpectedly, by its nonmetabolizable analog 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor. These findings reveal a previously unknown function of glucose as a signaling molecule that protects cells from death induced by plasma membrane depolarization, independently of its role in glycolysis. Components of this cancer cell death pathway represent potential therapeutic targets against cancer.

Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation.

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.

Axonal targeting of the serotonin transporter in cultured rat dorsal raphe neurons is specified by SEC24C-dependent export from the endoplasmic reticulum.

Export of the serotonin transporter (SERT) from the endoplasmic reticulum (ER) is mediated by the SEC24C isoform of the coatomer protein-II complex. SERT must enter the axonal compartment and reach the presynaptic specialization to perform its function, i.e., the inward transport of serotonin. Refilling of vesicles is contingent on the operation of an efficient relay between SERT and the vesicular monoamine transporter-2 (VMAT2). Here, we visualized the distribution of both endogenously expressed SERT and heterologously expressed variants of human SERT in dissociated rat dorsal raphe neurons to examine the role of SEC24C-dependent ER export in axonal targeting of SERT. We conclude that axonal delivery of SERT is contingent on recruitment of SEC24C in the ER. This conclusion is based on the following observations. (1) Both endogenous and heterologously expressed SERT were delivered to the extensive axonal arborizations and accumulated in bouton-like structures. (2) In contrast, SERT-(607)RI(608)-AA, in which the binding site of SEC24C is disrupted, remained confined to the microtubule-associated protein 2-positive somatodendritic compartment. (3) The overexpression of dominant-negative SEC24C-D(796)V/D(797)N (but not of the corresponding SEC24D mutant) redirected both endogenous SERT and heterologously expressed yellow fluorescent protein-SERT from axons to the somatodendritic region. (4) SERT-K(610)Y, which harbors a mutation converting it into an SEC24D client, was rerouted from the axonal to the somatodendritic compartment by dominant-negative SEC24D. In contrast, axonal targeting of the VMAT2 was disrupted by neither dominant-negative SEC24C nor dominant-negative SEC24D. This suggests that SERT and VMAT2 reach the presynaptic specialization by independent routes.

Budding yeast greatwall and endosulfines control activity and spatial regulation of PP2A(Cdc55) for timely mitotic progression.

Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2A(Cdc55) phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2A(Cdc55) in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2A(Cdc55) activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2A(Cdc55), timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2A(Cdc55), which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.

Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation.

Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The conserved Atg5-Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5-Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5-Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre-autophagosomal structure but is essential for autophagy and cytoplasm-to-vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5-Atg12/Atg16 complex during autophagosome formation.

M-Track: detecting short-lived protein-protein interactions in vivo.

We developed a protein-proximity assay in yeast based on fusing a histone lysine methyltransferase onto a bait and its substrate onto a prey. Upon binding, the prey is stably methylated and detected by methylation-specific antibodies. We applied this approach to detect varying interaction affinities among proteins in a mitogen-activated protein kinase pathway and to detect short-lived interactions between protein phosphatase 2A and its substrates that have so far escaped direct detection.

Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells.

Neuritic alterations are a major feature of many neurodegenerative disorders. Methylation of protein phosphatase 2A (PP2A) catalytic C subunit by the leucine carboxyl methyltransferase (LCMT1), and demethylation by the protein phosphatase methylesterase 1, is a critical PP2A regulatory mechanism. It modulates the formation of PP2A holoenzymes containing the Bα subunit, which dephosphorylate key neuronal cytoskeletal proteins, including tau. Significantly, we have reported that LCMT1, methylated C and Bα expression levels are down-regulated in Alzheimer disease-affected brain regions. In this study, we show that enhanced expression of LCMT1 in cultured N2a neuroblastoma cells, which increases endogenous methylated C and Bα levels, induces changes in F-actin organization. It promotes serum-independent neuritogenesis and development of extended tau-positive processes upon N2a cell differentiation. These stimulatory effects can be abrogated by LCMT1 knockdown and S-adenosylhomocysteine, an inhibitor of methylation reactions. Expression of protein phosphatase methylesterase 1 and the methylation-site L309Δ C subunit mutant, which decrease intracellular methylated C and Bα levels, block N2a cell differentiation and LCMT1-mediated neurite formation. Lastly, inducible and non-inducible knockdown of Bα in N2a cells inhibit process outgrowth. Altogether, our results establish a novel mechanistic link between PP2A methylation and development of neurite-like processes.

A phosphorylation switch regulates the transcriptional activation of cell cycle regulator p21 by histone deacetylase inhibitors.

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells.