Regulation of Proapoptotic Mammalian ste20–Like Kinase MST2 by the IGF1-Akt Pathway

Background

Hippo, a Drosophila serine/threonine kinase, promotes apoptosis and restricts cell growth and proliferation. Its mammalian homolog MST2 has been shown to play similar role and be regulated by Raf-1 via a kinase-independent mechanism and by RASSF family proteins through forming complex with MST2. However, regulation of MST2 by cell survival signal remains largely unknown.

Methodology/Principal Findings

Using immunoblotting, in vitro kinase and in vivo labeling assays, we show that IGF1 inhibits MST2 cleavage and activation induced by DNA damage through the phosphatidylinosotol 3-kinase (PI3K)/Akt pathway. Akt phosphorylates a highly conserved threonine-117 residue of MST2 in vitro and in vivo, which leads to inhibition of MST2 cleavage, nuclear translocation, autophosphorylation-Thr180 and kinase activity. As a result, MST2 proapoptotic and growth arrest function was significantly reduced. Further, inverse correlation between pMST2-T117/pAkt and pMST2-T180 was observed in human breast tumors.

Conclusions/Significance

Our findings demonstrate for the first time that extracellular cell survival signal IGF1 regulates MST2 and that Akt is a key upstream regulator of MST2.     

Molecular implication of PP2A and Pin1 in the Alzheimer's disease specific hyperphosphorylation of Tau

Background

Tau phosphorylation and dephosphorylation regulate in a poorly understood manner its physiological role of microtubule stabilization, and equally its integration in Alzheimer disease (AD) related fibrils. A specific phospho-pattern will result from the balance between kinases and phosphatases. The heterotrimeric Protein Phosphatase type 2A encompassing regulatory subunit PR55/Bα (PP2A_T55α) is a major Tau phosphatase in vivo, which contributes to its final phosphorylation state. We use NMR spectroscopy to determine the dephosphorylation rates of phospho-Tau by this major brain phosphatase, and present site-specific and kinetic data for the individual sites including the pS202/pT205 AT8 and pT231 AT180 phospho-epitopes.

Methodology/Principal Findings

We demonstrate the importance of the PR55/Bα regulatory subunit of PP2A within this enzymatic process, and show that, unexpectedly, phosphorylation at the pT231 AT180 site negatively interferes with the dephosphorylation of the pS202/pT205 AT8 site. This inhibitory effect can be released by the phosphorylation dependent prolyl cis/trans isomerase Pin1. Because the stimulatory effect is lost with the dimeric PP2A core enzyme (PP2A_D) or with a phospho-Tau T231A mutant, we propose that Pin1 regulates the interaction between the PR55/Bα subunit and the AT180 phospho-epitope on Tau.

Conclusions/Significance

Our results show that phosphorylation of T231 (AT180) can negatively influence the dephosphorylation of the pS202/pT205 AT8 epitope, even without an altered PP2A pool. Thus, a priming dephosphorylation of pT231 AT180 is required for efficient PP2A_T55α-mediated dephosphorylation of pS202/pT205 AT8. The sophisticated interplay between priming mechanisms reported for certain Tau kinases and the one described here for Tau phosphatase PP2A_T55α may contribute to the hyperphosphorylation of Tau observed in AD neurons.     

Defects of protein phosphatase 2A causes corticosteroid insensitivity in severe asthma

Background

Corticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma.

Methodology/Principal Findings

Corticosteroid sensitivity was determined by the dexamethasone ability to inhibit TNFα-induced IL-8 or LPS-induced TNFα production. PP2A expression, glucocorticoid receptor (GR) nuclear translocation defined as the nuclear/cytoplasmic GR ratio and phosphorylation of GR-Ser²²⁶, c-Jun N-terminal kinase 1 (JNK1) and PP2A were analysed by Western-blotting. Phosphatase activity was measured by fluorescence-based assay. Okadaic acid (OA), a PP2A inhibitor, reduced corticosteroid sensitivity with reduced GR nuclear translocation and increased GR phosphorylation in U937 monocytic cells. PP2A knockdown by RNA interference showed similar effects. IL-2/IL-4 treatment to U937 reduced corticosteroid sensitivity, and PP2A expression/activity. In peripheral blood mononuclear cells (PBMCs) from severe asthma, the PP2A expression and activity were significantly reduced with concomitant enhancement of PP2A_C-Tyr³⁰⁷ phosphorylation compared with those in healthy volunteers. As the results, GR-Ser²²⁶ and JNK1 phosphorylation were increased. The expression and activity of PP2A were negatively correlated with phosphorylation levels of GR-Ser²²⁶. Furthermore, co-immunoprecipitation assay in U937 cells revealed that PP2A associated with GR and JNK1 and IL-2/IL-4 exposure caused dissociation of each molecule. Lastly, PP2A overexpression increased corticosteroid sensitivity in U937 cells.

Conclusions/Significance

PP2A regulates GR nuclear translocation and corticosteroid sensitivity possibly by dephosphorylation of GR-Ser²²⁶ via dephosphorylation of upstream JNK1. This novel mechanism will provide new insight for the development of new therapy for severe asthma.     

Activation of protein kinase C delta following cerebral ischemia leads to release of cytochrome C from the mitochondria via bad pathway

Background

The release of cytochrome c from the mitochondria following cerebral ischemia is a key event leading to cell death. The goal of the present study was to determine the mechanisms involved in post-ischemic activation of protein kinase c delta (δPKC) that lead to cytochrome c release.

Methods/Findings

We used a rat model of cardiac arrest as an in vivo model, and an in vitro analog, oxygen glucose deprivation (OGD) in rat hippocampal synaptosomes. Cardiac arrest triggered translocation of δPKC to the mitochondrial fraction at 1 h reperfusion. In synaptosomes, the peptide inhibitor of δPKC blocked OGD-induced translocation to the mitochondria. We tested two potential pathways by which δPKC activation could lead to cytochrome c release: phosphorylation of phospholipid scramblase-3 (PLSCR3) and/or protein phosphatase 2A (PP2A). Cardiac arrest increased levels of phosphorlyated PLSCR3; however, inhibition of δPKC translocation failed to affect the OGD-induced increase in PLSCR3 in synaptosomal mitochondria suggesting the post-ischemic phosphorylation of PLSCR3 is not mediated by δPKC. Inhibition of either δPKC or PP2A decreased cytochrome c release from synaptosomal mitochondria. Cardiac arrest results in the dephosphorylation of Bad and Bax, both downstream targets of PP2A promoting apoptosis. Inhibition of δPKC or PP2A prevented OGD-induced Bad, but not Bax, dephosphorylation. To complement these studies, we used proteomics to identify novel mitochondrial substrates of δPKC.

Conclusions

We conclude that δPKC initiates cytochrome c release via phosphorylation of PP2A and subsequent dephosphorylation of Bad and identified δPKC, PP2A and additional mitochondrial proteins as potential therapeutic targets for ischemic neuroprotection.     

Specific Targeting of Caspase-9/PP2A Interaction as Potential New Anti-Cancer Therapy

Purpose

PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo.

Experimental Design

We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitonealy administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated.

Results

We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed.

Conclusion

Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression.     

Structural Comparison of Human Mammalian Ste20-Like Kinases

Background

The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains.

Principal Findings

We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family.

Significance

The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design.

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PP2A Mediated AMPK Inhibition Promotes HSP70 Expression in Heat Shock Response

Background

Under stress, AMP-activated protein kinase (AMPK) plays a central role in energy balance, and the heat shock response is a protective mechanism for cell survival. The relationship between AMPK activity and heat shock protein (HSP) expression under stress is unclear.

Methodology/Principal Findings

We found that heat stress induced dephosphorylation of AMPKα subunit (AMPKα) in various cell types from human and rodent. In HepG2 cells, the dephosphorylation of AMPKα under heat stress in turn caused dephosphorylation of acetyl-CoA carboxylase and upregulation of phosphoenolpyruvate carboxykinase, two downstream targets of AMPK, confirming the inhibition of AMPK activity by heat stress. Treatment of HepG2 cells with phosphatase 2A (PP2A) inhibitor okadaic acid or inhibition of PP2A expression by RNA interference efficiently reversed heat stress-induced AMPKα dephosphorylation, suggesting that heat stress inhibited AMPK through activation of PP2A. Heat stress- and other HSP inducer (CdCl₂, celastrol, MG132)-induced HSP70 expression could be inhibited by AICAR, an AMPK specific activator. Inhibition of AMPKα expression by RNA interference reversed the inhibitory effect of AICAR on HSP70 expression under heat stress. These results indicate that AMPK inhibition under stress contribute to HSP70 expression. Mechanistic studies showed that activation of AMPK by AICAR had no effect on heat stress-induced HSF1 nuclear translocation, phosphorylation and binding with heat response element in the promoter region of HSP70 gene, but significantly decreased HSP70 mRNA stability.

Conclusions/Significance

These results demonstrate that during heat shock response, PP2A mediated AMPK inhibition upregulates HSP70 expression at least partially through stabilizing its mRNA, which suggests a novel mechanism for HSP induction under stress.     

Dopaminergic Neuronal Imaging in Genetic Parkinson's Disease: Insights into Pathogenesis

Objectives

To compare the dopaminergic neuronal imaging features of different subtypes of genetic Parkinson''s Disease.

Methods

A retrospective study of genetic Parkinson''s diseases cases in which DaTSCAN (123I-FP-CIT) had been performed. Specific non-displaceable binding was calculated for bilateral caudate and putamen for each case. The right:left asymmetry index and striatal asymmetry index was calculated.

Results

Scans were available from 37 cases of monogenetic Parkinson''s disease (7 glucocerebrosidase (GBA) mutations, 8 alpha-synuclein, 3 LRRK2, 7 PINK1, 12 Parkin). The asymmetry of radioligand uptake for Parkinson''s disease with GBA or LRRK2 mutations was greater than that for Parkinson''s disease with alpha synuclein, PINK1 or Parkin mutations.

Conclusions

The asymmetry of radioligand uptake in Parkinsons disease associated with GBA or LRRK2 mutations suggests that interactions with additional genetic or environmental factors may be associated with dopaminergic neuronal loss.     

Pharmacologic modulation of serine/threonine phosphorylation highly sensitizes PHEO in a MPC cell and mouse model to conventional chemotherapy

Background

The failure of cytotoxic cancer regimens to cure the most drug-resistant, well-differentiated solid tumors has been attributed to the heterogeneity of cell types that differ in their capacities for growth, differentiation, and metastases. We investigated the effect of LB1, a small molecule inhibitor of serine/threonine protein phosphatase 2A (PP2A), on its ability to inhibit a low growth fraction and highly drug-resistant solid neuroendocrine tumor, such as metastatic pheochromocytoma (PHEO). Subsequently, we evaluated the increased efficacy of chemotherapy combined with LB1.

Methodology/Principal Findings

The effect of LB1 and temozolomide (TMZ), a standard chemotherapeutic agent that alone only transiently suppressed the growth and regression of metastatic PHEO, was evaluated in vitro on a single PHEO cell line and in vivo on mouse model of metastatic PHEO. In the present study, we show that metastatic PHEO, for which there is currently no cure, can be eliminated by combining LB1, thereby inhibiting PP2A, with TMZ. This new treatment approach resulted in long term, disease-free survival of up to 40% of animals bearing multiple intrahepatic metastases, a disease state that the majority of patients die from. Inhibition of PP2A was associated with prevention of G1/S phase arrest by p53 and of mitotic arrest mediated by polo-like kinase 1 (Plk-1).

Conclusions/Significance

The elimination of DNA damage-induced defense mechanisms, through transient pharmacologic inhibition of PP2A, is proposed as a new approach for enhancing the efficacy of non-specific cancer chemotherapy regimens against a broad spectrum of low growth fraction tumors very commonly resistant to cytotoxic drugs.     

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern

Background

Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome.

Results

PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y’s phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism.

Conclusions

In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-384) contains supplementary material, which is available to authorized users.