Peter Storz

Reactive oxygen species in cancer

Abstract Elevated rates of reactive oxygen species (ROS) have been detected in almost all cancers, where they promote many aspects of tumour development and progression. However, tumour cells also express increased levels of antioxidant proteins to detoxify from ROS, suggesting that a delicate balance of intracellular ROS levels is required for cancer cell function. Further, the radical generated, the location of its generation, as well as the local concentration is important for the cellular functions of ROS in cancer. A challenge for novel therapeutic strategies will be the fine tuning of intracellular ROS signalling to effectively deprive cells from ROS-induced tumour promoting events, towards tipping the balance to ROS-induced apoptotic signalling. Alternatively, therapeutic antioxidants may prevent early events in tumour development, where ROS are important. However, to effectively target cancer cells specific ROS-sensing signalling pathways that mediate the diverse stress-regulated cellular functions need to be identified. This review discusses the generation of ROS within tumour cells, their detoxification, their cellular effects, as well as the major signalling cascades they utilize, but also provides an outlook on their modulation in therapeutics.

Reactive oxygen species in tumor progression

The generation of reactive oxygen radicals in mammalian cells profoundly affects numerous critical cellular functions, and the absence of efficient cellular detoxification mechanisms which remove these radicals can result in several human diseases. Growing evidence suggests that reactive oxygen species (ROS) within cells act as second messengers in intracellular signaling cascades which induce and maintain the oncogenic phenotype of cancer cells. ROS are tumorigenic by virtue of their ability to increase cell proliferation, survival, cellular migration, and also by inducing DNA damage leading to genetic lesions that initiate tumorigenicity and sustain subsequent tumor progression. However, it is also known that ROS can induce cellular senescence and cell death and can therefore function as anti-tumorigenic agents. Therefore, the mechanisms by which cells respond to reactive oxygen species depends on the molecular background of cell and tissues, the location of ROS production and the concentration of individual ROS species. Carcinoma cells produce ROS at elevated rates in vitro, and in vivo many tumors appear persistent to oxidative stress. Thus, the finding that a diet rich in antioxidants or the elimination of ROS by antioxidant compounds prevents the development of certain cancers provided the setting for subsequent investigation of the tumorigenic actions of reactive oxygen species. This review outlines the current knowledge on the various roles of ROS in tumor development and progression.

PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid.

Tumor necrosis factor (TNF‐alpha) stimulates a number of signal transduction pathways in which phospholipases produce lipid second messengers. However, the immediate molecular targets of these messengers, in particular those of ceramide and arachidonic acid (AA) and their role in TNF signaling are not well defined. In this study we investigated the relationship of ceramide and AA in regulating an atypical PKC isozyme, PKC zeta. U937 cells responding to TNF‐alpha treatment with NF kappa B activation displayed enhanced phosphorylation of PKC zeta, which is already detectable 30 s after stimulation. [14C]ceramide specifically binds to and regulates kinase activity of PKC zeta in a biphasic manner. Binding studies indicate high and low affinity binding with bmax values of 60 and 600 nM and Kd values of 7.5 and 320 nM respectively. At ceramide concentrations as low as 0.5 nM an up to 4‐fold increase in autophosphorylation is obtained, which, at concentrations > 60 nM, again declines to basal levels. Interestingly, AA competes for ceramide binding and inhibits basal and ceramide‐stimulated PKC zeta kinase activity at < 100 nM. Metabolism of [14C]ceramide in cells is slow and is inhibited in the presence of equimolar concentrations of lyso‐phosphatidylcholine. Based on the bifunctional modulation of PKC zeta by the lipid messengers ceramide and AA, a model of TNF signal pathways is suggested in which PKC zeta takes a central position, acting as a molecular switch between mitogenic and growth inhibitory signals of TNF‐alpha.

A rapid method for determining protein kinase phosphorylation specificity

Selection of target substrates by protein kinases is strongly influenced by the amino acid sequence surrounding the phosphoacceptor site. Identification of the preferred peptide phosphorylation motif for a given kinase permits the production of efficient peptide substrates and greatly simplifies the mapping of phosphorylation sites in protein substrates. Here we describe a combinatorial peptide library method that allows rapid generation of phosphorylation motifs for serine/threonine kinases.

Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIβ at the Golgi complex

Protein kinase D (PKD) regulates the fission of vesicles originating from the trans-Golgi network. We show that phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) — a key player in the structure and function of the Golgi complex — is a physiological substrate of PKD. Of the three PKD isoforms, only PKD1 and PKD2 phosphorylated PI4KIIIβ at a motif that is highly conserved from yeast to humans. PKD-mediated phosphorylation stimulated lipid kinase activity of PI4KIIIβ and enhanced vesicular stomatitis virus G-protein transport to the plasma membrane. The identification of PI4KIIIβ as one of the PKD substrates should help to reveal the molecular events that enable transport-carrier formation.

Protein kinase D mediates a stress‐induced NF‐κB activation and survival pathway

The activation of the transcription factor NF‐κB is critical for a number of physiological responses. Here, we provide evidence for a signaling pathway that mediates NF‐κB activation in response to oxidative stress. We show that tyrosine phosphorylation of protein kinase D (PKD) at Y463 in the Pleckstrin Homology (PH) domain is mediated by the Src and Abl tyrosine kinase signaling pathway, and that this is both necessary and sufficient to activate NF‐κB in response to oxidative stress. PKD activates NF‐κB through the IKK complex and more specifically, IKKβ, leading to IκBα degradation. We also present evidence that this pathway is required for increased cellular survival in response to oxidative stress. We propose a model in which protection from oxidative stress‐induced cell death requires the tyrosine phosphorylation of PKD leading to the activation of the transcription factor NF‐κB.

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

ABSTRACT 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 PKCε 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.

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

ABSTRACT 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.

Protein kinase D1 regulates cofilin-mediated F-actin reorganization and cell motility through slingshot

Dynamic actin remodelling processes at the leading edge of migrating tumour cells are concerted events controlled by a fine-tuned temporal and spatial interplay of kinases and phosphatases. Actin severing is regulated by actin depolymerizing factor (ADF)/cofilin, which regulates stimulus-induced lamellipodia protrusion and directed cell motility. Cofilin is activated by dephosphorylation through phosphatases of the slingshot (SSH) family. SSH activity is strongly increased by its binding to filamentous actin (F-actin); however, other upstream regulators remain unknown. Here we show that in response to RhoA activation, protein kinase D1 (PKD1) phosphorylates the SSH enzyme SSH1L at a serine residue located in its actin-binding motif. This generates a 14-3-3-binding motif and blocks the localization of SSH1L to F-actin-rich structures in the lamellipodium by sequestering it in the cytoplasm. Consequently, expression of constitutively active PKD1 in invasive tumour cells enhanced the phosphorylation of cofilin and effectively blocked the formation of free actin-filament barbed ends and directed cell migration.

A Phosphorylation State-specific Antibody Recognizes Hsp27, a Novel Substrate of Protein Kinase D

The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser82 in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.