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Biomedical subjects

Peter Storz

Publications and source records attributed to Peter Storz.

18 recordsLinked to original sources
PubMed2006

Mitochondrial ROS--radical detoxification, mediated by protein kinase D.

The mitochondrial electron transport chain is the major source for the production of oxygen radicals. Mitochondria-generated reactive oxygen species (mROS) have been implicated in decreasing the life span and contributing to age-related diseases (known as the free radical theory of aging). Recently, the serine/threonine kinase protein kinase D1 (PKD1) was identified as a mitochondrial sensor for oxidative stress. mROS-activated PKD regulates a radical-sensing signaling pathway, which relays mROS production to the induction of nuclear genes that mediate cellular detoxification and survival. This PKD regulated signaling pathway is the first known mitochondria located and mitochondrially regulated antioxidant system that protects these organelles and cells from oxidative stress-mediated damage or cell death. The identification of this and further intracellular protective signaling pathways provides an opportunity to manipulate the effects of mROS, and might provide the key to targeting aging effects and age-related diseases that have been linked to mitochondrial dysfunctions.

Animals↗
PubMed2006

Reactive oxygen species-mediated mitochondria-to-nucleus signaling: a key to aging and radical-caused diseases.

Mitochondria-generated reactive oxygen species have been implicated as a common feature that connects aging of organisms and age-related diseases. Efficient elimination of these radicals by antioxidants correlates with increased life span. Understanding how the mitochondrion signals to the nucleus to regulate antioxidant proteins might be a key to aging processes and treatment of human diseases.

Aging↗
PubMed2005

Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIbeta 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 IIIbeta (PI4KIIIbeta) - 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 PI4KIIIbeta at a motif that is highly conserved from yeast to humans. PKD-mediated phosphorylation stimulated lipid kinase activity of PI4KIIIbeta and enhanced vesicular stomatitis virus G-protein transport to the plasma membrane. The identification of PI4KIIIbeta as one of the PKD substrates should help to reveal the molecular events that enable transport-carrier formation.

1-Phosphatidylinositol 4-Kinase↗
PubMed2005

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.

Animals↗
PubMed2005

Functional dichotomy of A20 in apoptotic and necrotic cell death.

ROS (reactive oxygen species) play important roles in the progression of a number of human pathologies. ROS promote cell death, but can also induce gene transcription. The transcription factor NF-kappaB (nuclear factor kappaB) plays a critical role in oxidative stress responses. One of the proteins regulated by NF-kappaB is the zinc-finger protein A20. In TNF (tumour necrosis factor)-alpha signalling, NF-kappaB induction of A20 leads to increased cell survival. In the present paper, we show that in response to oxidative stress, A20 actually enhances cell death by necrosis, but not by apoptosis. Exposure of cells to ROS leads to the up-regulation of A20 which acts via a negative-feedback loop to block NF-kappaB activation and cellular survival. Silencing of A20 by RNAi (RNA interference) increases both the induction of NF-kappaB and the subsequent survival of cells exposed to high doses of oxidative stress, which, in untreated cells, promotes death by necrosis. Cells which express high basal levels of A20 are less protected from oxidative-stress-induced cell death when compared with cells with lower A20 expression. We also show that A20 regulates NF-kappaB by blocking the degradation of IkappaB (inhibitory protein kappaB) alpha. These data highlight a novel role for A20 in oxidative stress responses by terminating NF-kappaB-dependent survival signalling and thus sensitizing cells to death by necrosis.

Apoptosis↗
PubMed2005

Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H2O2 but not tumor necrosis factor.

Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Go6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Go6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Go6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Go6976 but not by Go6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling.

14-3-3 Proteins↗
PubMed2005

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 Ser(82) 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.

Amino Acid Motifs↗
PubMed2005

Anti-CD63 antibodies suppress IgE-dependent allergic reactions in vitro and in vivo.

High-affinity IgE receptor (FcepsilonRI) cross-linking on mast cells (MCs) induces secretion of preformed allergy mediators (degranulation) and synthesis of lipid mediators and cytokines. Degranulation produces many symptoms of immediate-type allergic reactions and is modulated by adhesion to surfaces coated with specific extracellular matrix (ECM) proteins. The signals involved in this modulation are mostly unknown and their contribution to allergic reactions in vivo is unclear. Here we report the generation of monoclonal antibodies that potently suppress FcepsilonRI-induced degranulation, but not leukotriene synthesis. We identified the antibody target as the tetraspanin CD63. Tetraspanins are membrane molecules that form multimolecular complexes with a broad array of molecules including ECM protein-binding beta integrins. We found that anti-CD63 inhibits MC adhesion to fibronectin and vitronectin. Furthermore, anti-CD63 inhibits FcepsilonRI-mediated degranulation in cells adherent to those ECM proteins but not in nonadherent cells. Thus the inhibition of degranulation by anti-CD63 correlates with its effect on adhesion. In support of a mechanistic linkage between the two types of inhibition, anti-CD63 had no effect on FcepsilonRI-induced global tyrosine phosphorylation and calcium mobilization but impaired the Gab2-PI3K pathway that is known to be essential for both degranulation and adhesion. Finally, we showed that these antibodies inhibited FcepsilonRI-mediated allergic reactions in vivo. These properties raise the possibility that anti-CD63 could be used as therapeutic agents in MC-dependent diseases.

Adaptor Proteins, Signal Transducing↗
PubMed2005

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-kappaB 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.

Alleles↗
PubMed2004

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

Activation of the inducible transcription factor nuclear factor kappaB (NF-kappaB) occurs in cells exposed to oxidative stress, and the serine/threonine kinase protein kinase D (PKD) is critical for signal relay to NF-kappaB. We have recently delineated two coordinated events that 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 protein kinase C (PKC) isoform PKCdelta. The result is fully active PKD that controls NF-kappaB activation through the IkappaB kinase (IKK) complex. Here, we investigate the mechanism by which PKD controls IKK/NF-kappaB activation. Resveratrol, a potent antioxidant, blocks both PKD activation and NF-kappaB induction. In particular, resveratrol blocked PKD activation loop phosphorylation and activity, and this was caused by a specific inhibition of the Ser738/Ser742 kinase PKCdelta. On the other hand, 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-kappaB 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 antioxidant, resveratrol specifically blocks oxidative stress-dependent NF-kappaB activation by interfering with PKD phosphorylation and association with the IKK complex.

Alleles↗
PubMed2004

Hepatocyte resistance to oxidative stress is dependent on protein kinase C-mediated down-regulation of c-Jun/AP-1.

The prevention of injury from reactive oxygen species is critical for cellular resistance to many death stimuli. Resistance to death from the superoxide generator menadione in the hepatocyte cell line RALA255-10G is dependent on down-regulation of the c-Jun N-terminal kinase (JNK)/AP-1 signaling pathway by extracellular signal-regulated kinase 1/2 (ERK1/2). Because protein kinase C (PKC) regulates both oxidant stress and JNK signaling, the ability of PKC to modulate hepatocyte death from menadione through effects on AP-1 was examined. PKC inhibition with Ro-31-8425 or bisindolylmaleimide I sensitized this cell line to death from menadione. Menadione treatment led to activation of PKCmicro, or protein kinase D (PKD), but not PKCalpha/beta, PKCzeta/lambda, or PKCdelta/. Menadione induced phosphorylation of PKD at Ser-744/748, but not Ser-916, and translocation of PKD to the nucleus. PKC inhibition blocked menadione-induced phosphorylation of PKD, and expression of a constitutively active PKD prevented death from Ro-31-8425/menadione. PKC inhibition led to a sustained overactivation of JNK and c-Jun in response to menadione as determined by in vitro kinase assay and immunoblotting for the phosphorylated forms of both proteins. Cell death from PKC inhibition and menadione treatment resulted from c-Jun activation, since death was blocked by adenoviral expression of the c-Jun dominant negative TAM67. PKC and ERK1/2 independently down-regulated JNK/c-Jun, since inhibition of either kinase failed to affect activation of the other kinase, and simultaneous inhibition of both pathways caused additive JNK/c-Jun activation and cell death. Resistance to death from superoxide therefore requires both PKC/PKD and ERK1/2 activation in order to down-regulate proapoptotic JNK/c-Jun signaling.

Animals↗
PubMed2004

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.

Amino Acid Motifs↗
PubMed2004

Protein kinase Cdelta selectively regulates protein kinase D-dependent activation of NF-kappaB in oxidative stress signaling.

Protein kinase D (PKD) participates in activation of the transcription factor NF-kappaB (nuclear factor kappaB) 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-kappaB 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 PKCdelta (protein kinase Cdelta), a kinase that is activated by Src in response to oxidative stress. We also show that other PKCs, including PKCepsilon and PKCzeta, do not participate in PKD activation or NF-kappaB 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-PKCdelta pathway. Once active, the signal is relayed to the activation of NF-kappaB in oxidative stress responses.

Enzyme Activation↗
PubMed2003

Tyrosine phosphorylation of protein kinase D in the pleckstrin homology domain leads to activation.

Protein kinase D (PKD) is a member of the AGC family of Ser/Thr kinases and is distantly related to protein kinase C (PKC). Formerly known as PKCmu, PKD contains protein domains not found in conventional PKC isoforms. A functional pleckstrin homology (PH) domain is critical for the regulation of PKD activity. Here we report that PKD is tyrosine-phosphorylated within the PH domain, leading to activation. This phosphorylation is mediated by a pathway that consists of the Src and Abl tyrosine kinases and occurs in response to stimulation with pervanadate and oxidative stress. Mutational analysis revealed three tyrosine phosphorylation sites (Tyr(432), Tyr(463), and Tyr(502)), which are regulated by the Src-Abl pathway, and phosphorylation of only one of these (Tyr(463)) leads to PKD activation. By using a phospho-specific antibody, we show that Abl directly phosphorylates PKD at Tyr(463) in vitro, and in cells phosphorylation of this site is sufficient to mediate full activation of PKD. Mutation of the other two sites, Tyr(432) and Tyr(502), had no significant influence on PKD activity. These data reveal a tyrosine phosphorylation-dependent activation mechanism for PKD and suggest that this event contributes to the release of the autoinhibitory PKD PH domain leading to kinase activation and downstream responses.

Amino Acid Sequence↗
PubMed2003

Protein kinase D mediates a stress-induced NF-kappaB activation and survival pathway.

The activation of the transcription factor NF-kappaB is critical for a number of physiological responses. Here, we provide evidence for a signaling pathway that mediates NF-kappaB 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-kappaB in response to oxidative stress. PKD activates NF-kappaB through the IKK complex and more specifically, IKKbeta, leading to I(kappa)B(alpha) 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-kappaB.

3T3 Cells↗
PubMed2002

3'-phosphoinositide-dependent kinase-1 (PDK-1) in PI 3-kinase signaling.

The recently discovered 3'-phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase which phosphorylates several members of the conserved AGC kinase superfamily (comprising the prototypes protein kinases A (PKA), G (PKG) and C (PKC)). Phosphorylation of a threonine or serine residue in the activation loop (also known as the T-loop) of these kinases is a critical step in their activation, and is typically accompanied by additional phosphorylations elsewhere in the molecule. Phosphorylation of the activation loop is a common regulatory mechanism shared by most serine/threonine as well as tyrosine kinases as it facilitates alignment of amino acid residues in the active site which are involved in the phosphotransferase reaction. Therefore the discovery of PDK-1 as the enzyme which mediates this event in many protein kinases introduced a new and important step in signaling pathways which regulate numerous important cellular processes including cellular survival, glucose transport and metabolism, tumor progression as well as protein translation. Moreover, the finding that PDK-1 function is mediated in part by the phosphoinositide 3'-OH-kinase (PI 3-K) pathway also provided an explanation as to how the lipid products of PI 3-K, namely phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol-3,4-5-trisphosphate (PtdIns-3,4,5-P3) stimulate the activation of protein kinase-dependent signaling pathways. These initial landmark observations were followed by many important studies which provided additional mechanistic insight into both PDK-1 regulation as well as the role of this kinase in cellular function. This review will focus on the regulation of PDK-1 and the various mechanisms which it uses to contribute to the activation of target kinases.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2001

Protein kinase C (PKC)eta-mediated PKC mu activation modulates ERK and JNK signal pathways.

Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKC mu/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKC mu/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKC mu partially colocalizes with PKC eta in different cell types. Colocalization depends on the presence of the PKC mu pleckstrin homology domain. Coexpression of constitutively active PKC eta with PKC mu leads to a significant enhancement of the PKC mu substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser(738/742) of PKC mu, whereas Ser(910) autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKC eta directly phosphorylates PKC mu on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKC eta-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKC mu/PKC eta affecting different arms of the p38/ERK/JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKC mu for interaction with and activation by PKC eta as pleckstrin homology domain and activation loop.

Breast Neoplasms↗