Protein Kinase D1 Maintains the Epithelial Phenotype by Inducing a DNA-Bound, Inactive SNAI1 Transcriptional Repressor Complex

Protein kinase D1 is downregulated in its expression in invasive ductal carcinoma of the breast and in invasive breast cancer cells, but its functions in normal breast epithelial cells is largely unknown. The epithelial phenotype is maintained by cell-cell junctions formed by E-cadherin. In cancer cells loss of E-cadherin expression contributes to an invasive phenotype. This can be mediated by SNAI1, a transcriptional repressor for E-cadherin that contributes to epithelial-to-mesenchymal transition (EMT).Here we show that PKD1 in normal murine mammary gland (NMuMG) epithelial cells is constitutively-active in its basal state and prevents a transition to a mesenchymal phenotype. Investigation of the involved mechanism suggested that PKD1 regulates the expression of E-cadherin at the promoter level through direct phosphorylation of the transcriptional repressor SNAI1. PKD1-mediated phosphorylation of SNAI1 occurs in the nucleus and generates a nuclear, inactive DNA/SNAI1 complex that shows decreased interaction with its co-repressor Ajuba. Analysis of human tissue samples with a newly-generated phosphospecific antibody for PKD1-phosphorylated SNAI1 showed that regulation of SNAI1 through PKD1 occurs in vivo in normal breast ductal tissue and is decreased or lost in invasive ductal carcinoma.Our data describe a mechanism of how PKD1 maintains the breast epithelial phenotype. Moreover, they suggest, that the analysis of breast tissue for PKD-mediated phosphorylation of SNAI1 using our novel phosphoS11-SNAI1-specific antibody may allow predicting the invasive potential of breast cancer cells

Insulin selectively activates STAT5b, but not STAT5a, via a JAK2-independent signalling pathway in Kym-1 rhabdomyosarcoma cells

AbstractThe STAT multigene family of transcriptional regulators conveys signals from several cytokines and growth factors upon phosphorylation by janus kinases (JAK). Activation of STAT5 is typically mediated by JAK2, but more recent data indicate a direct activation by the insulin receptor kinase. STAT5 exists in two closely homologous isoforms, STAT5a and b. We here describe the selective tyrosine phosphorylation of STAT5b in Kym-1 cells in response to insulin. Blocking insulin signalling by HNMPA-(AM)3, an insulin receptor kinase inhibitor, resulted in the loss of insulin-induced STAT5b tyrosine phosphorylation, whereas the inhibition of JAK2 by the JAK selective inhibitor tyrphostin AG490 had no effect. By contrast, in the same cells, IFNγ-induced STAT5b activation was JAK2-dependent, indicating that this signal pathway is functional in Kym-1 cells. We conclude from this rhabdomyosarcoma model that STAT5b, but not STAT5a is a direct target of the insulin receptor kinase

Protein Kinase Cδ Selectively Regulates Protein Kinase D-Dependent Activation of NF-κB in Oxidative Stress Signaling

Protein kinase D (PKD) participates in activation of the transcription factor NF-κB (nuclear factor κB) in cells exposed to oxidative stress, leading to increased cellular survival. We previously demonstrated that phosphorylation of PKD at Tyr463 in the PH (pleckstrin homology) domain is mediated by the Src-Abl pathway and that it is necessary for PKD activation and subsequent NF-κB induction. Here we show that activation of PKD in response to oxidative stress requires two sequential signaling events, i.e., phosphorylation of Tyr463 by Abl, which in turn promotes a second step, phosphorylation of the PKD activation loop (Ser738/Ser742). We show that this is mediated by PKCδ (protein kinase Cδ), a kinase that is activated by Src in response to oxidative stress. We also show that other PKCs, including PKCɛ and PKCζ, do not participate in PKD activation or NF-κB induction. We propose a model in which two coordinated signaling events are required for PKD activation. Tyrosine phosphorylation in the PH domain at Tyr463, mediated by the Src-Abl pathway, which in turn facilitates the phosphorylation of Ser738/Ser742 in the activation loop, mediated by the Src-PKCδ pathway. Once active, the signal is relayed to the activation of NF-κB in oxidative stress responses

Activation loop phosphorylation controls protein kinase D-dependent activation of nuclear factor

modified Eagle's medium; IKK, IκB kinase; LPS, lipopolysaccharide; MBP, myelin basic protein; NF-κB, nuclear factor κ-B; PH, pleckstrin homology; PKD, protein kinase D; PKC, protein kinase C; PMA, 12-phorbol 13-myristate acetate; TBS, Tris-buffered saline; TNF-α, tumor necrosis factor-α. MOL#687 3 ABSTRACT Activation of the inducible transcription factor NF-κB (Nuclear Factor κ-B) occurs in cells exposed to oxidative stress, and the serine/threonine kinase PKD (protein kinase D) is critical for signal relay to NF-κB. We have recently delineated two coordinated events which control PKD activation in response to oxidative stress, phosphorylation at Tyr463 by the tyrosine kinase Abl, and phosphorylation at the activation loop Ser738/Ser742 by the PKC isoform PKCδ. The result is fully active PKD which controls NF-κB activation through the IKK complex. Here, we investigate the mechanism by which PKD controls IKK/NF-κB activation. Resveratrol, a potent anti-oxidant, blocks both PKD activation and NF-κB induction. Specifically, resveratrol blocked PKD activation loop phosphorylation and activity, and this was due to a specific inhibition of the Ser738/Ser742 kinase, PKCδ. Conversely, resveratrol did not affect Abl kinase activity and had no effect on Tyr463 phosphorylation. Moreover, we show that the mechanism by which resveratrol inhibits NF-κB is by blocking the translocation of PKD to the IKK complex, specifically by inhibiting Ser738/Ser742 phosphorylation. We therefore propose that rather than acting as an anti-oxidant, resveratrol specifically blocks oxidative stressdependent NF-κB activation by interfering with PKD phosphorylation and association with the IKK complex

Protein Kinase D Mediates Mitochondrion-to-Nucleus Signaling and Detoxification from Mitochondrial Reactive Oxygen Species

Efficient elimination of mitochondrial reactive oxygen species (mROS) correlates with increased cellular survival and organism life span. Detoxification of mitochondrial ROS is regulated by induction of the nuclear SOD2 gene, which encodes the manganese-dependent superoxide dismutase (MnSOD). However, the mechanisms by which mitochondrial oxidative stress activates cellular signaling pathways leading to induction of nuclear genes are not known. Here we demonstrate that release of mROS activates a signal relay pathway in which the serine/threonine protein kinase D (PKD) activates the NF-κB transcription factor, leading to induction of SOD2. Conversely, the FOXO3a transcription factor is dispensable for mROS-induced SOD2 induction. PKD-mediated MnSOD expression promotes increased survival of cells upon release of mROS, suggesting that mitochondrion-to-nucleus signaling is necessary for efficient detoxification mechanisms and cellular viability

Downregulation of TRAF2 mediates NIK-induced pancreatic cancer cell proliferation and tumorigenicity.

Increased levels of NF-κB are hallmarks of pancreatic ductal adenocarcinoma (PDAC) and both classical and alternative NF-κB activation pathways have been implicated.Here we show that activation of the alternative pathway is a source for the high basal NF-κB activity in PDAC cell lines. Increased activity of the p52/RelB NF-κB complex is mediated through stabilization and activation of NF-κB-inducing kinase (NIK). We identify proteasomal downregulation of TNF receptor-associated factor 2 (TRAF2) as a mechanism by which levels of active NIK are increased in PDAC cell lines. Such upregulation of NIK expression and activity levels relays to increased proliferation and anchorage-independent growth, but not migration or survival of PDAC cells.Rapid growth is one characteristic of pancreatic cancer. Our data indicates that the TRAF2/NIK/NF-κB2 pathway regulates PDAC cell tumorigenicity and could be a valuable target for therapy of this cancer

Generation of Hydrogen Peroxide and Downstream Protein Kinase D1 Signaling Is a Common Feature of Inducers of Pancreatic Acinar-to-Ductal Metaplasia

Pancreatic acinar-to-ductal metaplasia (ADM) is a reversible process that occurs after pancreatic injury, but becomes permanent and leads to pancreatic lesions in the presence of an oncogenic mutation in KRAS,. While inflammatory macrophage-secreted chemokines, growth factors that activate epidermal growth factor receptor (EGFR) and oncogenic KRAS have been implicated in the induction of ADM, it is currently unclear whether a common underlying signaling mechanism exists that drives this process. In this study, we show that different inducers of ADM increase levels of hydrogen peroxide, most likely generated at the mitochondria, and upregulate the expression of Protein Kinase D1 (PKD1), a kinase that can be activated by hydrogen peroxide. PKD1 expression in acinar cells affects their survival and mediates ADM, which is in part due to the PKD1 target NF-κB. Overall, our data implicate ROS-PKD1 signaling as a common feature of different inducers of pancreatic ADM

NIK is a critical regulator of transformed growth in PDAC cells.

<p><b>A:</b> Panc1 or MiaPaca2 cells (5×10⁵ cells, 6 cm dishes) stably expressing control (scrambled) shRNA or NIK-shRNA (two different sequences, NIK-shRNA1 or NIK-shRNA2) were subjected to soft agar colony formation assays. A fraction of the transfected cells were collected and analyzed for NIK expression using Western blot analysis and antibodies directed against NIK (anti-NIK) or β-actin (anti-β-actin) as loading control. The asterisks indicate statistical significance. Scale bars represent 1 mm. <b>B:</b> Panc1 cells (5×10⁵ cells, 6 cm dishes) were lentivirally infected with control virus or virus for expression of constitutively active NIK (NIK.T559D mutant) and subjected to soft agar colony formation assays. Before seeding a fraction of the cells were lysed and analyzed for expressed active NIK using Western blot and antibodies directed against FLAG-tagged NIK.T559D (anti-FLAG) or β-actin (anti-β-actin) as a loading control. The asterisk indicates statistical significance. Scale bars represent 1 mm. <b>C:</b> Panc1 cells (5×10⁵ cells, 6 cm dishes) stably expressing control (scrambled) shRNA, NIK-shRNA1 or NIK-shRNA2 (top row), or cells lentivirally infected with control virus or NIK.T559D mutant (bottom row) were seeded in 3D Matrigel culture. At days 10 (shRNA cells) or 14 (NIK.T559D expressing cells) after seeding colony growth was analyzed by ImagePro. The bar represents 500 µm.</p