Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes

Synthesis and biological evaluation of dialkylsubstituted maleic anhydrides as novel inhibitors of Cdc25 dual specificity phosphatases

International audienc

Phylogenetically Distant Viruses Use the Same BH3-Only Protein Puma to Trigger Bax/Bak-Dependent Apoptosis of Infected Mouse and Human Cells

<div><p>Viruses can trigger apoptosis of infected host cells if not counteracted by cellular or viral anti-apoptotic proteins. These protective proteins either inhibit the activation of caspases or they act as Bcl-2 homologs to prevent Bax/Bak-mediated outer mitochondrial membrane permeabilization (MOMP). The exact mechanism by which viruses trigger MOMP has however remained enigmatic. Here we use two distinct types of viruses, a double stranded DNA virus, herpes simplex virus-1 (HSV-1) and a positive sense, single stranded RNA virus, Semliki Forest virus (SFV) to show that the BH3-only protein Puma is the major mediator of virus-induced Bax/Bak activation and MOMP induction. Indeed, when Puma was genetically deleted or downregulated by shRNA, mouse embryonic fibroblasts and IL-3-dependent monocytes as well as human colon carcinoma cells were as resistant to virus-induced apoptosis as their Bax/Bak double deficient counterparts (Bax/Bak-/-). Puma protein expression started to augment after 2 h postinfection with both viruses. Puma mRNA levels increased as well, but this occurred after apoptosis initiation (MOMP) because it was blocked in cells lacking Bax/Bak or overexpressing Bcl-x_L. Moreover, none of the classical Puma transcription factors such as p53, p73 or p65 NFκB were involved in HSV-1-induced apoptosis. Our data suggest that viruses use a Puma protein-dependent mechanism to trigger MOMP and apoptosis in host cells.</p></div

HSV-1-induced caspase-3 activation and apoptosis requires Puma and to a smaller extent Bmf.

<p><b>(A)</b> Annexin-V/PI FACS analysis of SV40 TAg WT, Bmf-/-, Bik-/-, Bad-/-, Bid-/- and 3T9-immortalized WT, Puma-/-, Bim-/- and Noxa-/- MEFs infected with 10 moi HSV-1 for 0, 24 or 48 h (hpi). <b>(B)</b> Caspase-3/-7 (DEVDase) activity assay and <b>(C)</b> anti-caspase-3 (pro-caspase-3 and cleaved caspase-3) western blot analysis of total extracts of 3T9 WT and Puma-/- MEFs infected with HSV-1 for 0, 14, 24 or 48 h. Anti-actin as loading control in (C). <b>(D)</b> Annexin-V/PI FACS analysis of puromycin selected, mixed populations of SV40 TAg-transformed and 3T9-immortalized MEFs infected with lentiviruses carrying either a scrambled shRNA (sh-Ctrl) or a shRNA of mouse Puma (sh-Puma), infected with HSV-1 for 0, 24, 48 or 72 h (hpi). The 3T9 WT and Puma-/- cells are shown as controls. Data in (A), (B) and (D) are the means of at least three independent experiments using two different clones of WT and each knock-out cell line in (A) and (B) and mixed populations in (D) ± SEM. The <i>p</i> values are the following: (A) Bmf-/- versus WT: <i>p</i> = 0.005 for 24 h, <i>p</i> = 0.01 for 48 h; Puma-/- versus WT: <i>p</i> < 0.001 for 24 h and 48 h, n = 6. (B) Puma-/- versus WT: <i>p</i> < 0.001 for 14, 24 h and 48 h, n = 5. (D) sh-Puma versus sh-Ctrl and Puma-/- versus WT: <i>p</i> < 0.001 for 24 h, 48 h and 72 h, n = 4.</p

HSV-1-induced apoptosis of U937 monocytes depends on Bax/Bak and is most efficient when NFκB activation if prevented.

<p><b>(A)</b> Annexin-V/PI FACS analysis of human U937 monocytes carrying the pcDNA3 vector or expressing a dominant-negative version of IκBα (mIκBα) (which prevents NFκB activation), infected with 50 moi of herpes simplex virus-1 (HSV-1) in the presence or absence of 25 μM of the general caspase inhibitor QVD for 0, 24 and 48 h. The number of cells lacking annexin-V/PI staining (the lower left quadrants in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126645#pone.0126645.s001" target="_blank">S1 Fig</a>) are depicted. They represent cells which are protected against both apoptotic and necroptosis/necrotic cell death. <b>(B)</b> Anti-Bax and anti-Bak western blot analysis of total extracts of puromycin-selected, mixed population U937 mIκBα cells infected with lentivirus carrying a scrambled shRNA (sh-Ctrl) or shRNAs of human Bax (sh-Bax), Bak (sh-Bak) or both. Anti-actin as loading control. <b>(C)</b> Annexin-V/PI FACS analysis of the cell lines described in (B) after infecting them with 50 moi of HSV-1 for 0, 24 or 48 h. Data in (A) and (C) are the means of at least three independent experiments ± SEM. The <i>p</i> values are the following: (A) mIκBα versus pcDNA3: <i>p</i> = 0.008 for 24 h, <i>p</i> = 0.003 for 48 h; mIκBα + QVD versus mIκBα - QVD: <i>p</i> = 0.01 for 24 h, <i>p</i> = 0.005 for 48 h, n = 4. (C) sh-Bax + sh-Bak versus sh-Ctrl, <i>p</i> < 0.001 for 24 and 48 h; sh-Bax or sh-Bak versus sh-Ctrl, not significant, n = 5. hpi: hours post infection. kD: kilo Dalton.</p

HSV-1-induced caspase-3 activation and apoptosis of SV40 TAg MEFs are predominantly mediated via Bax/Bak.

<p><b>(A)</b> Caspase-3/-7 activity (DEVDase) assay and <b>(B)</b> anti-caspase-3 (pro-caspase-3 and cleaved caspase-3) western blots of total extracts as well as <b>(C)</b> annexin-V/PI FACS analysis of SV40 TAg WT and Bax/Bak-/- MEFs infected with 10 moi of HSV-1 for 0 (mock), 14, 24 or 48 h (hpi) in the absence or presence of 25 μM QVD. The number of cells lacking annexin-V/PI staining (the lower left quadrants in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126645#pone.0126645.s001" target="_blank">S1 Fig</a>) are depicted in (C). Anti-actin as loading control in (B). Data in (A) and (C) are the means of at least three independent experiments using three different clones of WT and Bax/Bak-/- cells ± SEM. The <i>p</i> values are the following: (A) Bax/Bak-/- versus WT cells: <i>p</i> < 0.001 for 24 and 48 h, n = 5. (C) Bax/Bak-/- versus WT cells: <i>p</i> < 0.001 for 24 and 48 h; WT + QVD versus WT—QVD: <i>p</i> = 0.005 for 24 h, <i>p</i> = 0.01 for 48 h; Bax/Bak-/- + QVD versus Bax/Bak-/-—QVD: <i>p</i> = 0.05 for 24 h, <i>p</i> = 0.03 for 48 h, n = 5.</p

Puma knock-down in MEFs and its knock-out in FDMs also markedly diminish SFV-induced caspase-3 activation and apoptosis.

<p><b>(A)</b> Caspase-3/-7 (DEVDase) activity assay of total extracts of puromycin selected, mixed populations of SV40 TAg-transformed and 3T9-immortalized MEFs infected with lentiviruses carrying either a scrambled shRNA (sh-Ctrl) or a shRNA of mouse Puma (sh-Puma), infected with SFV for 0, 6, 14 or 24 h (hpi). As a control the data of 3T9 Puma-/- MEFs are shown. In addition, the caspase activities are compared to those from cells treated with 10 ng/ml FasL for 14 h. Data are the means of at least three independent experiments ± SEM. The <i>p</i> values are the following: 3T9 sh-Puma versus 3T9 sh-Ctrl: <i>p</i> = 0.008 for 6 h, <i>p</i> < 0.001 for 14 and 24 h; SV40 TAg sh-Puma versus SV40 TAg sh-Ctrl and 3T9 Puma-/- versus 3T9 WT: <i>p</i> < 0.001 for 6, 14 and 24 h, n = 4. <b>(B)</b> Annexin-V/PI FACS analysis of WT, Puma-/- and Bax/Bax-/- FDM cells infected with 10 moi of SFV for 0, 14, 24 or 36 h (hpi). Data are the means of at least three independent experiments using three different clones of WT, Puma-/- and Bax/Bak-/- cells ± SEM. The <i>p</i> values are the following: Puma-/- versus WT: <i>p</i> = 0.03 for 14 h, <i>p</i> < 0.001 for 24 and 36 h. Bax/Bak-/- versus WT: <i>p</i> = 0.01 for 14 h, <i>p</i> < 0.001 for 24 and 36 h, n = 4.</p

Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes

SFV enhances Puma mRNA and protein levels in MEFs and FDM cells, but mRNA increase is late and Bax/Bak-dependent.

<p><b>(A)</b> Quantitative/real time reverse transcriptase PCR (qRT-PCR) of Puma mRNA isolated from SV40 TAg WT and Bax/Bak-/- MEFs infected with 10 moi of SFV for 0, 1, 2, 4, 6, 8, 10, 14, 18 or 24 h (hpi). The mRNA values were normalized to the ribosomal housekeeping 18S gene and depicted as 2^-∆∆Ct relative to mock cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126645#sec009" target="_blank">Materials and Methods</a> for details). Data are the means of at least three independent experiments ± SEM. The <i>p</i> values are the following: SFV-treated WT versus untreated: <i>p</i> = 0.005 for 6 h, <i>p</i> = 0.01 for 8 h, <i>p</i> = 0.008 for 10 h. SFV-treated Bax/Bak-/- versus untreated: not significant, n = 5. <b>(B)</b> Anti-Puma, anti-caspase-3 (pro-caspase-3 and cleaved caspase-3) and anti-PARP western blot analysis of total cell extracts of SV40 TAg WT MEFs infected with SFV for 0, 2, 6, 8, 10 or 18 h. Anti-actin as loading control.</p