PP2A Regulatory Subunit Research Articles

PAK6-mediated phosphorylation of PPP2R2C regulates LRRK2-PP2A complex formation.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson's disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2's phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation.

Microcystin-LR, a Cyanobacterial Toxin, Induces Changes in the Organization of Membrane Compartments in Arabidopsis

To evaluate the effects of the cyanobacterial toxin microcystin-LR (MCY-LR, a protein phosphatase inhibitor) and diquat (DQ, an oxidative stress inducer) on the organization of tonoplast, the effect of MCY-LR on plastid stromule formation and on mitochondria was investigated in wild-type Arabidopsis. Tonoplast was also studied in PP2A catalytic (c3c4) and regulatory subunit mutants (fass-5 and fass-15). These novel studies were performed by CLSM microscopy. MCY-LR is produced during cyanobacterial blooms. The organization of tonoplast of PP2A mutants of Arabidopsis is much more sensitive to MCY-LR and DQ treatments than that of wild type. In c3c4, fass-5 and fass-15, control and treated plants showed increased vacuole fragmentation that was the strongest when the fass-5 mutant was treated with MCY-LR. It is assumed that both PP2A/C and B" subunits play an important role in normal formation and function of the tonoplast. In wild-type plants, MCY-LR affects mitochondria. Under the influence of MCY-LR, small, round-shaped mitochondria appeared, while long/fused mitochondria were typical in control plants. Presumably, MCY-LR either inhibits the fusion of mitochondria or induces fission. Consequently, PP2A also plays an important role in the fusion of mitochondria. MCY-LR also increased the frequency of stromules appearing on chloroplasts after 1 h treatments. Along the stromules, signals can be transported between plastids and endoplasmic reticulum. It is probable that they promote a faster response to stress.

Kin-3 genetically suppresses sur-6 in centrosome assembly during Caenorhabditis elegans embryogenesis.

Protein phosphorylation plays a critical role in cell cycle progression. In Caenorhabditis elegans , Casein Kinase II (CK2) negatively regulates centrosome assembly, and Protein Phosphatase 2A (PP2A) SUR-6 /B55 acts as a positive regulator of centrosome duplication, suggesting CK2 and PP2A SUR-6 /B55 play opposing roles in centrosome assembly. Here, we examined the genetic interaction between kin-3 , encoding the catalytic subunit of CK2, and sur-6 , encoding the PP2Aregulatory subunit SUR-6 /B55, in Caenorhabditis elegans embryos. Our results show that kin-3 (RNAi) partially restores normal centrosome duplication and embryonic viability to hypomorphic sur-6 mutants, suggesting that kin-3 genetically suppresses sur-6 in centrosome assembly during Caenorhabditis elegans embryogenesis.

Clinical and molecular characteristics of a novel rare de novo variant in PPP2CA in a patient with a developmental disorder, autism, and epilepsy.

PP2A-related (neuro) developmental disorders are a family of genetic diseases caused by a heterozygous alteration in one of several genes encoding a subunit of type 2A protein phosphatases. Reported affected genes, so far, are PPP2R5D, encoding the PP2A regulatory B56δ subunit; PPP2R1A, encoding the scaffolding Aα subunit; and PPP2CA, encoding the catalytic Cα subunit-in that order of frequency. Patients with a pathogenic de novo mutation in one of these genes, in part, present with overlapping features, such as generalized hypotonia, intellectual and developmental delay, facial dysmorphologies, seizures, and autistic features, and, in part, with opposite features, e.g., smaller versus larger head sizes or normal versus absent corpus callosum. Molecular variant characterization has been consistent so far with loss-of-function or dominant-negative disease mechanisms for all three affected genes. Here, we present a case report of another PPP2CA-affected individual with a novel de novo missense variant, resulting in a one-amino acid substitution in the Cα subunit: p.Cys196Arg. Biochemical characterization of the variant revealed its pathogenicity, as it appeared severely catalytically impaired, showed mildly affected A subunit binding, and moderately decreased binding to B/B55, B"/PR72, and all B56 subunits, except B56γ1. Carboxy-terminal methylation appeared unaffected, as was binding to B"'/STRN3-all being consistent with a partial loss of function. Clinically, the girl presented with mild-to-moderate developmental delay, a full-scale IQ of 83, mild dysmorphic facial features, tonic-clonic seizures, and autistic behaviors. Brain MRI appeared normal. We conclude that this individual falls within the milder end of the clinical and molecular spectrum of previously reported PPP2CA cases.

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107.

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine protein phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R.' Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether, these data provide key insights into PP2A/B55α's mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α's role in multiple cellular processes.

Tumor-activatable biomineralized nanotherapeutics for integrative glucose starvation and sensitized metformin therapy

Metformin is a clinically-approved anti-diabetic drug with emerging antitumor potential, but its antitumor activity is highly susceptible to local glucose abundance. Herein, we construct a nanotherapeutic platform based on biocompatible constituents to sensitize tumor cells for metformin therapy via cooperative glucose starvation. The nanoplatform was synthesized through the spontaneous biomineralization of glucose oxidase (GOx) and metformin in amorphous calcium phosphate nanosubstrate, which was further modified with polyethylene glycol and cRGD ligands. This biomineralized nanosystem could efficiently deliver the therapeutic payloads to tumor cells in a targeted and bioresponsive manner. Here GOx could catalyze the oxidation of glucose into gluconic acid and H2O2, thus depleting the glucose in tumor intracellular compartment while accelerating the release of the entrapped therapeutic payloads. The selective glucose deprivation would not only disrupt tumor energy metabolism, but also upregulate the PP2A regulatory subunit B56δ and sensitize tumor cells to the metformin-induced CIP2A inhibition, leading to efficient apoptosis induction via PP2A-GSK3β-MCL-1 axis with negligible side effects. This study may offer new avenues for targeted tumor therapy in the clinical context.

MRBLE-pep Measurements Reveal Accurate Binding Affinities for B56, a PP2A Regulatory Subunit.

Signal transduction pathways rely on dynamic interactions between protein globular domains and short linear motifs (SLiMs). The weak affinities of these interactions are essential to allow fast rewiring of signaling pathways and downstream responses but also pose technical challenges for interaction detection and measurement. We recently developed a technique (MRBLE-pep) that leverages spectrally encoded hydrogel beads to measure binding affinities between a single protein of interest and 48 different peptide sequences in a single small volume. In prior work, we applied it to map the binding specificity landscape between calcineurin and the PxIxIT SLiM (Nguyen, H. Q. et al. Elife 2019, 8). Here, using peptide sequences known to bind the PP2A regulatory subunit B56α, we systematically compare affinities measured by MRBLE-pep or isothermal calorimetry (ITC) and confirm that MRBLE-pep accurately quantifies relative affinity over a wide dynamic range while using a fraction of the material required for traditional methods such as ITC.

Abstract 1986: p53/FBXL20 axis negatively regulates the protein stability of PR55α, a PP2A regulatory subunit

Abstract PP2A holoenzymes are the majority of Ser/Thr phosphatases in human cells and each PP2A complex contains one scaffold subunit, one catalytic subunit, and one regulatory subunit. PP2A regulatory subunits, existing in four sub-families encoded by 15 genes, determine the substrate specificities and intracellular localizations of PP2A. We previously reported an essential role for PR55α, a PP2A regulatory subunit, in the support of malignant potential of pancreatic cancer cells, which includes promoting YAP-activation that supports anchorage-independent growth and metastasis. Furthermore, our data showed that PR55α level is elevated in pancreatic cancer relative to normal tissues and high PR55α levels correlate with poor patient survival. The studies in this report reveal a novel mechanism by which the p53 tumor suppressor negatively regulates PR55α protein stability. The results show that p53 inactivation by siRNA-knockdown, gene-deletion, HPV-E6 promoted degradation, or p53R175H mutant expression all results in the increase of PR55α expression. Subsequent studies demonstrate that this p53 function is mediated through its target gene FBXL20, a substrate recognition component of the SCF (Skp_Cullin_F-box) ubiquitin ligase complex that facilitates proteasomal degradation. As a result, the expression of mutant p53R175H in human pancreatic ductal cells mimics the PR55α effect, increasing c-Myc protein stability, and promoting anchorage-independent proliferation. Additionally, FBXL20 mRNA levels are significantly reduced in pancreatic cancers relative to normal control, and low FBXL20 levels correlate with poor patient survival, both of which inversely resemble the pathological effects of PR55α in pancreatic cancer. Collectively, these results define a novel p53 function in the suppression of PR55α expression. Citation Format: Ying Yan, Lepakshe Madduri, Nichole Brandquist, Chitra Palanivel, Sumin Zhou, Charles Enke, Michel Ouellette. p53/FBXL20 axis negatively regulates the protein stability of PR55α, a PP2A regulatory subunit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1986.

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

A disorder-related variant (E420K) of a PP2A-regulatory subunit (PPP2R5D) causes constitutively active AKT-mTOR signaling and uncoordinated cell growth

Functional genomic approaches have facilitated the discovery of rare genetic disorders and improved efforts to decipher their underlying etiology. PPP2R5D-related disorder is an early childhood onset condition characterized by intellectual disability, hypotonia, autism-spectrum disorder, macrocephaly, and dysmorphic features. The disorder is caused by de novo single nucleotide changes in PPP2R5D, which generate heterozygous dominant missense variants. PPP2R5D is known to encode a B’-type (B’56δ) regulatory subunit of a PP2A-serine/threonine phosphatase. To help elucidate the molecular mechanisms altered in PPP2R5D-related disorder, we used a CRISPR-single-base editor to generate HEK-293 cells in which a single transition (c.1258G>A) was introduced into one allele, precisely recapitulating a clinically relevant E420K variant. Unbiased quantitative proteomic and phosphoproteomic analyses of endogenously expressed proteins revealed heterozygous-dominant changes in kinase/phosphatase signaling. These data combined with orthogonal validation studies revealed a previously unrecognized interaction of PPP2R5D with AKT in human cells, leading to constitutively active AKT-mTOR signaling, increased cell size, and uncoordinated cellular growth in E420K-variant cells. Rapamycin reduced cell size and dose-dependently reduced RPS6 phosphorylation in E420K-variant cells, suggesting that inhibition of mTOR1 can suppress both the observed RPS6 hyperphosphorylation and increased cell size. Together, our findings provide a deeper understanding of PPP2R5D and insight into how the E420K-variant alters signaling networks influenced by PPP2R5D. Our comprehensive approach, which combines precise genome editing, isobaric tandem mass tag labeling of peptides generated from endogenously expressed proteins, and concurrent liquid chromatography–mass spectrometry (LC-MS3), also provides a roadmap that can be used to rapidly explore the etiologies of additional genetic disorders.

Phosphoproteomic Effects of Acute Depletion of PP2A Regulatory Subunit Cdc55.

Protein phosphatase regulatory subunits are increasingly recognized as promising drug targets. In the absence of an existing drug, inducible degradation provides a means of predicting candidate targets. Here auxin-inducible degradation of Saccharomyces cerevisiae PP2A regulatory subunit Cdc55 in combination with quantitative phosphoproteomics is employed. A prevalence of hyperphosphorylated phosphopeptides indicates that the approach successfully identified direct PP2ACdc55 targets. PRM follow up of data-dependent acquisition results confirmed that vacuolar amino acid transporters are among the proteins most strongly affected by Cdc55 depletion.

Cryo-EM structure of the deltaretroviral intasome in complex with the PP2A regulatory subunit B56\u03b3

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.

Abstract A44: PR55α regulatory subunit of PP2A inhibits the MOB1/LATS cascade and activates YAP in pancreatic cancer cells

Abstract PP2A holoenzyme complexes constitute the majority of Ser/Thr phosphatase activities in human cells. Each PP2A consists of a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (B). While the A and C subunits are highly conserved, a large number of B subunits share no homology, which determines PP2A substrate specificity and/or cellular localization. Although different PP2A complexes play distinct roles in cell signaling networks, PP2A has been defined only as a putative tumor suppressor, mostly based on loss-of-function studies using inhibitors (pharmacologic or biologic) for the highly conserved A or C subunit of PP2A. Many studies of specific pathways indicate that PP2A also possesses tumor-promoting function. We have reported an essential role of PR55α, a PP2A regulatory subunit, in supporting oncogenic phenotypes and in vivo tumorigenicity and metastasis of pancreatic cancer cells. In this study, we investigated a novel mechanism by which PR55α activates the YAP oncoprotein that plays an essential role in oncogenesis and metastasis of solid tumors including pancreatic cancer, resulting in its stabilization and nuclear retention. This mechanism involves PR55α’s inhibition of the MOB1-mediated LATS1/2 autoactivation, which otherwise would suppress YAP activation, and its direct activation of YAP itself. However, this mechanism does not involve the inhibition of upstream MST1/2 kinases and their subsequent T1079/T1041-phosphorylation of the LATS1/2 kinases. Furthermore, PR55α has the predominant effect on YAP activation, and the effect cannot be substituted by the depletion of either LATS1/2 or MOB1. Accordingly, PR55α is essential for YAP-promoted gene transcription, as well as for the anchorage-independent growth of cancer cells. In summary, current studies demonstrate a novel pancreatic cancer promoting mechanism through the PR55α-regulated PP2A. Citation Format: Ashley L. Hein, Nichole D. Brandquist, Caroline Y. Ouellette, Parthasarathy Seshacharyulu, Charles A. Enke, Michel M. Ouellette, Surinder K. Batra, Ying Yan. PR55α regulatory subunit of PP2A inhibits the MOB1/LATS cascade and activates YAP in pancreatic cancer cells [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A44.

B55α/PP2A Limits Endothelial Cell Apoptosis During Vascular Remodeling: A Complementary Approach To Disrupt Pathological Vessels?

How endothelial cells (ECs) migrate and form an immature vascular plexus has been extensively studied. Yet, mechanisms underlying vascular remodeling remain poorly established. A better understanding of these processes may lead to the design of novel therapeutic strategies complementary to current angiogenesis inhibitors. Starting from our previous observations that PP2A (protein phosphatase 2) regulates the HIF (hypoxia-inducible factor)/PHD-2 (prolyl hydroxylase 2)-constituted oxygen machinery, we hypothesized that this axis could play an important role during blood vessel formation, tissue perfusion, and oxygen restoration. We show that the PP2A regulatory subunit B55α is at the crossroad between vessel pruning and vessel maturation. Blood vessels with high B55α counter cell stress conditions and thrive for stabilization and maturation. When B55α is inhibited, ECs cannot cope with cell stress and undergo apoptosis, leading to massive pruning of nascent blood vessels. Mechanistically, we found that the B55α/PP2A complex restrains PHD-2 activity, promoting EC survival in a HIF-dependent manner, and furthermore dephosphorylates p38, altogether protecting ECs against cell stress occurring, for example, during the onset of blood flow. In tumors, EC-specific B55α deficiency induces pruning of immature-like tumor blood vessels resulting in delayed tumor growth and metastasis, without affecting nonpathological vessels. Consistently, systemic administration of a pan-PP2A inhibitor disrupts vascular network formation and tumor progression in vivo without additional effects on B55α-deficient vessels. Our data underline a unique role of the B55α/PP2A phosphatase complex in vessel remodeling and suggest the use of PP2A-inhibitors as potent antiangiogenic drugs targeting specifically nascent blood vessels with a mode-of-action complementary to VEGF-R (vascular endothelial growth factor receptor)-targeted therapies. Graphical Abstract: A graphical abstract is available for this article.

Role of the PP2A regulatory subunit B56α in regulation of cardiac responses to β-adrenergic stimulation

Role of the PP2A regulatory subunit B56α in regulation of cardiac responses to β-adrenergic stimulation

PR55\u03b1 regulatory subunit of PP2A inhibits the MOB1/LATS cascade and activates YAP in pancreatic cancer cells

PP2A holoenzyme complexes are responsible for the majority of Ser/Thr phosphatase activities in human cells. Each PP2A consists of a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (B). While the A and C subunits each exists only in two highly conserved isoforms, a large number of B subunits share no homology, which determines PP2A substrate specificity and cellular localization. It is anticipated that different PP2A holoenzymes play distinct roles in cellular signaling networks, whereas PP2A has only generally been defined as a putative tumor suppressor, which is mostly based on the loss-of-function studies using pharmacological or biological inhibitors for the highly conserved A or C subunit of PP2A. Recent studies of specific pathways indicate that some PP2A complexes also possess tumor-promoting functions. We have previously reported an essential role of PR55α, a PP2A regulatory subunit, in the support of oncogenic phenotypes, including in vivo tumorigenicity/metastasis of pancreatic cancer cells. In this report, we have elucidated a novel role of PR55α-regulated PP2A in the activation of YAP oncoprotein, whose function is required for anchorage-independent growth during oncogenesis of solid tumors. Our data show two lines of YAP regulation by PR55α: (1) PR55α inhibits the MOB1-triggered autoactivation of LATS1/2 kinases, the core member of the Hippo pathway that inhibits YAP by inducing its proteasomal degradation and cytoplasmic retention and (2) PR55α directly interacts with and regulates YAP itself. Accordingly, PR55α is essential for YAP-promoted gene transcriptions, as well as for anchorage-independent growth, in which YAP plays a key role. In summary, current findings demonstrate a novel YAP activation mechanism based on the PR55α-regulated PP2A phosphatase.

Integrin α5β1 regulates PP2A complex assembly through PDE4D in atherosclerosis.

Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site Ser651. Active PDE then hydrolyzes anti-inflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site in PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in athero-prone regions of arteries, and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also unexpectedly stabilized the PP2A-B55α complex. The integrin-regulated, pro-atherosclerotic transcription factor Yap is also dephosphorylated and activated through this pathway. PDE4D5 therefore mediates matrix-specific regulation of EC phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex with other targets. These results are likely to have widespread consequences for control of cell function by integrins.

A Conserved PP2A Regulatory Subunit Enforces Proportional Relationships Between Cell Size and Growth Rate.

Cell size is proportional to growth rate. Thus, cells growing rapidly in rich nutrients can be nearly twice the size of cells growing slowly in poor nutrients. This proportional relationship appears to hold across all orders of life, yet the underlying mechanisms are unknown. In budding yeast, most growth occurs during mitosis, and the proportional relationship between cell size and growth rate is therefore enforced primarily by modulating growth in mitosis. When growth is slow, the duration of mitosis is increased to allow more time for growth, yet the amount of growth required to complete mitosis is reduced, which leads to the birth of small daughter cells. Previous studies have found that Rts1, a member of the conserved B56 family of protein phosphatase 2A regulatory subunits, works in a TORC2 signaling network that influences cell size and growth rate. However, it was unclear whether Rts1 influences cell growth and size in mitosis. Here, we show that Rts1 is required for the proportional relationship between cell size and growth rate during mitosis. Moreover, nutrients and Rts1 influence the duration and extent of growth in mitosis via Wee1 and Pds1/securin, two conserved regulators of mitotic progression. Together, the data are consistent with a model in which global signals that set growth rate also set the critical amount of growth required for cell cycle progression, which would provide a simple mechanistic explanation for the proportional relationship between cell size and growth rate.

Proteomic characterization of MPK4 signaling network and putative substrates.

Combining genetic engineering of MPK4 activity and quantitative proteomics, we established an in planta system that enables rapid study of MPK4 signaling networks and potential substrate proteins. Mitogen activated protein kinase 4 (MPK4) is a multifunctional kinase that regulates various signaling events in plant defense, growth, light response and cytokinesis. The question of how a single protein modulates many distinct processes has spurred extensive research into the physiological outcomes resulting from genetic perturbation of MPK4. However, the mechanism by which MPK4 functions is still poorly understood due to limited data on the MPK4 networks including substrate proteins and downstream pathways. Here we introduce an experimental system that combines genetic engineering of kinase activity and quantitative proteomics to rapidly study the signaling networks of MPK4. First, we transiently expressed a constitutively active (MPK4CA) and an inactive (MPK4IN) version of a Brassica napus MPK4 (BnMPK4) in Nicotiana benthamiana leaves. Proteomics analysis revealed that BnMPK4 activation affects multiple pathways (e.g., metabolism, redox regulation, jasmonic acid biosynthesis and stress responses). Furthermore, BnMPK4 activation also increased protein phosphorylation in the phosphoproteome, from which putative MPK4 substrates were identified. Using protein kinase assay, we validated that a transcription factor TCP8-like (TCP8) and a PP2A regulatory subunit TAP46-like (TAP46) were indeed phosphorylated by BnMPK4. Taken together, we demonstrated the utility of proteomics and phosphoproteomics in elucidating kinase signaling networks and inidentification of downstream substrates.

Combination of Hypoglycemia and Metformin Impairs Tumor Metabolic Plasticity and Growth by Modulating the PP2A-GSK3β-MCL-1 Axis

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3β (GSK3β) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3β axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3β.