Chebulinic Acid Suppresses Adipogenesis in 3T3-L1 Preadipocytes by Inhibiting PPP1CB Activity

Depletion of protein phosphatase-1 catalytic subunit beta (PPP1CB), a serine/threonine protein phosphatase and potent adipogenic activator, suppresses the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Therefore, PPP1CB is considered as a potential therapeutic target for obesity. We screened 1033 natural products for PPP1CB inhibitors and identified chebulinic acid, which is abundantly present in the seeds of Euphoria longana and fruits of Terminalia chebula. Chebulinic acid strongly inhibited the hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate by PPP1CB (IC50 = 300 nM) and demonstrated potent antiadipogenic effects in 3T3-L1 preadipocytes in a concentration-dependent manner. Additional studies have demonstrated that chebulinic acid suppresses early differentiation by downregulating key transcription factors that control adipogenesis in 3T3-L1 cells. These results suggested that chebulinic acid may be a potential therapeutic agent for treating obesity by inhibiting PPP1CB activity.

The Oncogenic Potential of TCTEX1D4 is Modulated by the Phosphoprotein Phosphatase PP1

Protein phosphatase 1 (PP1) regulates several cellular events via interaction with multiple regulatory subunits. The human prostate proteome includes various PP1-interacting proteins; however, a very limited number of interactions is yet characterized and their role in prostate tumorigenesis remains poorly understood. Tctex1 domain-containing protein 4 (TCTEX1D4) was previously identified as a PP1-interacting protein, but its function, as well as the relevance of its interaction with PP1, are virtually unknown. In this study we addressed the role of the PP1/TCTEX1D4 complex in prostate tumorigenesis. We found distinct expression levels and subcellular distributions for TCTEX1D4 and PP1γ in human prostate epithelial normal-like and malignant cells. Moreover, we showed that TCTEX1D4 participates in the regulation of cell proliferation and modulation of microRNAs expression and that its interaction with PP1 controls its function. Taken together, our study provides first evidence for the involvement of the PP1/TCTEX1D4 complex in prostate tumorigenesis.

RIPK1 dephosphorylation and kinase activation by PPP1R3G/PP1γ promote apoptosis and necroptosis

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death. Many sites on RIPK1, including serine 25, are phosphorylated to inhibit its kinase activity and cell death. How these inhibitory phosphorylation sites are dephosphorylated is poorly understood. Using a sensitized CRISPR whole-genome knockout screen, we discover that protein phosphatase 1 regulatory subunit 3G (PPP1R3G) is required for RIPK1-dependent apoptosis and type I necroptosis. Mechanistically, PPP1R3G recruits its catalytic subunit protein phosphatase 1 gamma (PP1γ) to complex I to remove inhibitory phosphorylations of RIPK1. A PPP1R3G mutant which does not bind PP1γ fails to rescue RIPK1 activation and cell death. Furthermore, chemical prevention of RIPK1 inhibitory phosphorylations or mutation of serine 25 of RIPK1 to alanine largely restores cell death in PPP1R3G-knockout cells. Finally, Ppp1r3g−/− mice are protected from tumor necrosis factor-induced systemic inflammatory response syndrome, confirming the important role of PPP1R3G in regulating apoptosis and necroptosis in vivo.

Differential expression of protein phosphatase 1 regulatory inhibitor subunit 1A in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding protein phosphatase 1 regulatory inhibitor subunit 1A, PPP1R1A, when comparing primary tumors of the breast to the tissue of origin, the normal breast. PPP1R1A mRNA was present at significantly lower quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of PPP1R1A in primary tumors of the breast was correlated with overall survival in patients with basal-like and luminal A subtype cancer, demonstrating a relationship between primary tumor expression of a differentially expressed gene and patient survival outcomes influenced by PAM50 molecular subtype. PPP1R1A may be of relevance to initiation, maintenance or progression of cancers of the female breast.

The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction.

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of D. melanogaster KNL1 (Spc105) has never been shown to bind MTs nor to recruit PP1. Furthermore, the phospho-regulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. Here, these apparent discrepancies are resolved using in vitro and cell based-assays. A phospho-regulatory circuit, which utilizes Aurora B kinase (ABK), promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag, and deletion/chimera mutants are used to define the interplay of MT- and PP1-binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Core Control Principles of the Eukaryotic Cell Cycle

Cyclin dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different Cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs). However, the principles on which Cyclin-CDKs organise the temporal order of cell cycle events are contentious. The currently most widely accepted model, is that the S-CDKs and M-CDKs are functionally specialised, with significant different substrate specificities to execute different cell cycle events. A second model is that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall cellular CDK activity. Here we reconcile these two views of core cell cycle control. Using a multiplexed phosphoproteomics assay of in vivo S-CDK and M-CDK activities in fission yeast, we show that S-CDK and M-CDK substrate specificities are very similar, showing that S-CDKs are not completely specialised for S-phase alone. Normally S-CDK cannot undergo mitosis, but is able to do so when Protein Phosphatase 1 (PP1) is removed from the centrosome, allowing several mitotic substrates to be better phosphorylated by S-CDK in vivo. Thus, an increase in S-CDK activity in vivo is sufficient to allow S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine which temporally orders cell cycle progression is largely based upon a quantitative increase of CDK activity through the cell cycle, combined with minor qualitative differences in catalytic specialisation of S-CDKs and M-CDKs.

Protein Phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the Spindle Position Checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5 and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.

Phostensin Enables Lymphocyte Integrin Activation and Population of Peripheral Lymphoid Organs

Rap1 GTPase drives assembly of the Mig10/RIAM/lamellipodin–Integrin–Talin (MIT) complex that enables integrin dependent lymphocyte functions. Here we used tandem affinity tag based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (PTSN), a regulatory subunit of protein phosphatase 1, is a component of the complex. PTSN mediates dephosphorylation of Rap1 thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes PTSN, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18 null mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable to defective activation of integrins. Ppp1r18 null T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, PTSN enables lymphocyte integrin mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of PTSN ameliorates T cell mediated colitis.