Protein Phosphatase 2A Isoforms Utilizing Aβ Scaffolds Regulate Differentiation through Control of Akt Protein

Protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. It consists of a scaffolding A subunit to which a catalytic C subunit and one of many regulatory B subunits bind. Of the more than 80 PP2A isoforms, 10% use Aβ as a scaffold. This study demonstrates the isoform-specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K, and p53 in transformation. Comparisons of polyoma and SV40 small T antigens implicate Aβ in the control of differentiation. Knockdown of Aβ enhanced differentiation. Akt signaling regulated differentiation; its activation or inhibition promoted or blocked it, respectively. Aβ bound Akt. Enhancement of PP2A Aβ/Akt interaction by polyoma small T antigen increased turnover of Akt Ser-473 phosphorylation. Conversely, knockdown of Aβ promoted Akt activity and reduced turnover of phosphate at Ser-473 of Akt. These data provide new insight into the regulation of Akt, a protein of extreme importance in cancer. Furthermore, our results suggest that the role for Aβ in differentiation and perhaps tumor suppression may lie partly in its ability to negatively regulate Akt.     

Mutational Analysis of Glycogen Synthase Kinase 3β Protein Kinase Together with Kinome-Wide Binding and Stability Studies Suggests Context-Dependent Recognition of Kinases by the Chaperone Heat Shock Protein 90

The heat shock protein 90 (HSP90) and cell division cycle 37 (CDC37) chaperones are key regulators of protein kinase folding and maturation. Recent evidence suggests that thermodynamic properties of kinases, rather than primary sequences, are recognized by the chaperones. In concordance, we observed a striking difference in HSP90 binding between wild-type (WT) and kinase-dead (KD) glycogen synthase kinase 3β (GSK3β) forms. Using model cell lines stably expressing these two GSK3β forms, we observed no interaction between WT GSK3β and HSP90, in stark contrast to KD GSK3β forming a stable complex with HSP90 at a 1:1 ratio. In a survey of 91 ectopically expressed kinases in DLD-1 cells, we compared two parameters to measure HSP90 dependency: static binding and kinase stability following HSP90 inhibition. We observed no correlation between HSP90 binding and reduced stability of a kinase after pharmacological inhibition of HSP90. We expanded our stability study to >50 endogenous kinases across four cell lines and demonstrated that HSP90 dependency is context dependent. These observations suggest that HSP90 binds to its kinase client in a particular conformation that we hypothesize to be associated with the nucleotide-processing cycle. Lastly, we performed proteomics profiling of kinases and phosphopeptides in DLD-1 cells to globally define the impact of HSP90 inhibition on the kinome.     

Comparative Analysis of Gene Regulation by the Transcription Factor PPARα between Mouse and Human

Background

Studies in mice have shown that PPARα is an important regulator of hepatic lipid metabolism and the acute phase response. However, little information is available on the role of PPARα in human liver. Here we set out to compare the function of PPARα in mouse and human hepatocytes via analysis of target gene regulation.

Methodology/Principal Findings

Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARα agonist Wy14643 and gene expression profiling was performed using Affymetrix GeneChips followed by a systems biology analysis. Baseline PPARα expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 significantly induced the expression of 362–672 genes. Surprisingly minor overlap was observed between the Wy14643-regulated genes from mouse and human, although more substantial overlap was observed at the pathway level. Xenobiotics metabolism and apolipoprotein synthesis were specifically regulated by PPARα in human hepatocytes, whereas glycolysis-gluconeogenesis was regulated specifically in mouse hepatocytes. Most of the genes commonly regulated in mouse and human were involved in lipid metabolism and many represented known PPARα targets, including CPT1A, HMGCS2, FABP1, ACSL1, and ADFP. Several genes were identified that were specifically induced by PPARα in human (MBL2, ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4, Cd36, Ucp2, Pxmp4). Furthermore, several putative novel PPARα targets were identified that were commonly regulated in both species, including CREB3L3, KLF10, KLF11 and MAP3K8.

Conclusions/Significance

Our results suggest that PPARα activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARα as master regulator of hepatic lipid metabolism is generally well-conserved between mouse and human. Overall, however, PPARα regulates a mostly divergent set of genes in mouse and human hepatocytes.     

Disruption of Heat Shock Protein 90 (Hsp90)-Protein Kinase Cδ (PKCδ) Interaction by (?)-Maackiain Suppresses Histamine H1 Receptor Gene Transcription in HeLa Cells

The histamine H₁ receptor (H1R) gene is an allergic disease sensitive gene, and its expression level is strongly correlated with the severity of allergic symptoms. (−)-Maackiain was identified as a Kujin-derived anti-allergic compound that suppresses the up-regulation of the H1R gene. However, the underlying mechanism of H1R gene suppression remains unknown. Here, we sought to identify a target protein of (−)-maackiain and investigate its mechanism of action. A fluorescence quenching assay and immunoblot analysis identified heat shock protein 90 (Hsp90) as a target protein of (−)-maackiain. A pull-down assay revealed that (−)-maackiain disrupted the interaction of Hsp90 with PKCδ, resulting in the suppression of phorbol 12-myristate 13-acetate (PMA)-induced up-regulation of H1R gene expression in HeLa cells. Additional Hsp90 inhibitors, including 17-(allylamino)-17-demethoxygeldanamycin, celastrol, and novobiocin also suppressed PMA-induced H1R gene up-regulation. 17-(Allylamino)-17-demethoxygeldanamycin inhibited PKCδ translocation to the Golgi and phosphorylation of Tyr³¹¹ on PKCδ. These data suggest that (−)-maackiain is a novel Hsp90 pathway inhibitor. The underlying mechanism of the suppression of PMA-induced up-regulation of H1R gene expression by (−)-maackiain and Hsp90 inhibitors is the inhibition of PKCδ activation through the disruption of Hsp90-PKCδ interaction. Involvement of Hsp90 in H1R gene up-regulation suggests that suppression of the Hsp90 pathway could be a novel therapeutic strategy for allergic rhinitis.     

The Heat Shock Response Is Modulated by and Interferes with Toxic Effects of Scrapie Prion Protein and Amyloid β

The heat shock response (HSR) is an evolutionarily conserved pathway designed to maintain proteostasis and to ameliorate toxic effects of aberrant protein folding. We have studied the modulation of the HSR by the scrapie prion protein (PrP^Sc) and amyloid β peptide (Aβ) and investigated whether an activated HSR or the ectopic expression of individual chaperones can interfere with PrP^Sc- or Aβ-induced toxicity. First, we observed different effects on the HSR under acute or chronic exposure of cells to PrP^Sc or Aβ. In chronically exposed cells the threshold to mount a stress response was significantly increased, evidenced by a decreased expression of Hsp72 after stress, whereas an acute exposure lowered the threshold for stress-induced expression of Hsp72. Next, we employed models of PrP^Sc- and Aβ-induced toxicity to demonstrate that the induction of the HSR ameliorates the toxic effects of both PrP^Sc and Aβ. Similarly, the ectopic expression of cytosolic Hsp72 or the extracellular chaperone clusterin protected against PrP^Sc- or Aβ-induced toxicity. However, toxic signaling induced by a pathogenic PrP mutant located at the plasma membrane was prevented by an activated HSR or Hsp72 but not by clusterin, indicating a distinct mode of action of this extracellular chaperone. Our study supports the notion that different pathological protein conformers mediate toxic effects via similar cellular pathways and emphasizes the possibility to exploit the heat shock response therapeutically.     

Internalization by Cells and Antitumor Activity of Antibodies and Immunotoxins Specific for the Heat Shock Protein 90 β Isoform

Mouse monoclonal antibodies to Hsp90β (β isoform of heat shock protein 90) have been shown to bind specifically to Hsp90β localized on the surface of tumor and nontransformed cells. After binding to the membrane-associated Hsp90β, the antibodies actively dissociated into the culture medium and were also internalized by the cells. An immunoconjugate based on the Hsp90β-specific antibody and the cytotoxic agent mertansine did not have high cytotoxic activity for tumor cells in vitro. Administration of Hsp90β-specific antibodies in mice did not affect the growth of the primary Lewis lung carcinoma, while tumor metastasis to the lungs decreased and the average lifespan of mice increased. The results indicate a certain therapeutic potential of antibodies to Hsp90β for the treatment of tumor diseases.