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Alex Toker

Publications and source records attributed to Alex Toker.

At least 19 recordsLinked to original sources
PubMed2006

Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter.

Protein kinase D (PKD) regulates many diverse cellular functions in response to diacylglycerol. To monitor PKD signaling in live cells, we generated a genetically encoded fluorescent reporter for PKD activity, DKAR (D kinase activity reporter). DKAR expressed in mammalian cells undergoes reversible fluorescence resonance energy transfer changes upon activation and inhibition of endogenous PKD. Surprisingly, we find that agonist-evoked activation of PKD is driven not only by diacylglycerol production, but by Ca(2+). Furthermore, elevation of intracellular Ca(2+), in the absence of any other stimulus, is sufficient to activate PKD. Concurrent imaging of Ca(2+), diacylglycerol, and PKD activity reveals that thapsigargin-mediated elevation of intracellular Ca(2+) is closely followed by a robust increase in diacylglycerol production, in turn followed by PKD activation. The Ca(2+)-induced production of diacylglycerol and accompanying PKD activation is dependent on phospholipase C activity. These data reveal that Ca(2+) is a major contributor to the initiation of PKD signaling through positive feedback regulation of diacylglycerol production, unveiling a new mechanism in PKD activation.

Animals↗
PubMed2006

Akt signaling and cancer: surviving but not moving on.

The frequent deregulation of the phosphoinositide 3-kinase/Akt survival signaling pathway in cancer has prompted significant interest in blocking this pathway to treat cancer. Recently, however, two studies have shown that the Akt isoform Akt1 limits the invasive migration of breast cancer cells. These studies suggest that Akt1 may have a dual role in tumorigenesis, acting not only pro-oncogenically by suppressing apoptosis but also anti-oncogenically by suppressing invasion and metastasis. We discuss the possible implications of these findings for therapeutic development of Akt inhibitors to treat cancer.

Animals↗
PubMed2006

Akt/PKB signaling in cancer: a function in cell motility and invasion.

The PI 3-K signaling pathway has been the subject of intense investigation for over a decade because it regulates a multitude of cellular processes. Deregulation of this pathway often results in human diseases such as cancer. The serine/threonine kinase Akt, a major PI 3-K target in all cells, has received considerable attention because it provides tumor cells with enhanced survival capacity. Many tumors and tumor cells display elevated Akt protein expression and activity, and many of the proteins which regulate Akt itself are also often mutated in cancer. For this reason, Akt has become a viable drug target for cancer therapy. However, recent studies have shown that Akt can also function as a suppressor of tumor cell migration and invasion, phenotypes which are directly linked to metastatic tumor progression. Here we review these findings and consider the unexpected notion that Akt may function as a metastasis suppressor.

Animals↗
PubMed2006

NFAT induces breast cancer cell invasion by promoting the induction of cyclooxygenase-2.

The NFAT (nuclear factor of activated T cells) family of transcription factors plays a fundamental role in the transcriptional regulation of the immune response. However, NFATs are ubiquitously expressed, and recent evidence points to their important functions in human epithelial cells and carcinomas. Specifically, NFAT has been shown to be active in human breast and colon carcinoma cells and to promote their invasion through Matrigel. The mechanisms by which NFAT promotes invasion have not been defined. To identify NFAT target genes that induce carcinoma invasion, we have established stable breast cancer cell lines that inducibly express transcriptionally active NFAT. Gene expression profiling by cDNA microarray of cells induced to express NFAT revealed up-regulation of cyclooxygenase-2 (COX-2). Increased NFAT expression and activity induced COX-2 expression as well as prostaglandin E2 synthesis. This induction was more prominent when NFAT was activated by phorbol 12-myristate 13-acetate and calcium ionophore ionomycin and was blocked by the NFAT antagonist cyclosporin A. Breast cancer cells with elevated COX-2 expression showed increased invasion through Matrigel, and this was reduced in cells treated with COX-2 inhibitors. Conversely, loss of NFAT1 protein expression using small interfering RNA led to a reduction in COX-2 transcription and reduced invasion. Similarly, Matrigel invasion was reduced in cells in which COX-2 expression was reduced using specific siRNA. These findings demonstrate that NFAT promotes breast cancer cell invasion through the induction of COX-2 and the synthesis of prostaglandins.

Breast Neoplasms↗
PubMed2005

Akt blocks breast cancer cell motility and invasion through the transcription factor NFAT.

The phosphoinositide 3-kinase (PI 3-K) signaling axis is intimately associated with deregulated cancer cell growth, primarily by promoting increased survival through Akt/PKB (protein kinase B). However, there is relatively little information on the role of Akt in cancer cell motility, a key phenotype of invasive carcinomas. Here we report that activation of Akt inhibits carcinoma migration and invasion of breast cancer cells. Conversely, downregulation of Akt using RNA interference increased migration and invasion. Akt blunts invasion by inhibiting the transcriptional activity of NFAT (nuclear factor of activated T cells). Specifically, signaling through Akt reduces NFAT expression levels due to ubiquitination and proteasomal degradation, mediated by the E3 ubiquitin ligase HDM2. These results indicate that while Akt can promote tumor progression through increased cell survival mechanisms, it can block breast cancer cell motility and invasion by a mechanism that depends, at least in part, on the NFAT transcription factor.

Animals↗
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

A secreted form of ADAM9 promotes carcinoma invasion through tumor-stromal interactions.

Tumor cell invasion is a process regulated by integrins, matrix-degrading enzymes, and interactions with host tissue stromal cells. The ADAM family of proteins plays an important role in modulating various cellular responses. Here, we show that an alternatively spliced variant of ADAM9 is secreted by hepatic stellate cells and promotes carcinoma invasion. ADAM9-S induced a highly invasive phenotype in several human tumor cell lines in Matrigel assays, and the protease activity of ADAM9-S was required for invasion. ADAM9-S binds directly to alpha6beta4 and alpha2beta1 integrins on the surface of colon carcinoma cells through the disintegrin domain. ADAM9-S was also able to cleave laminin and promote invasion. Analysis of human liver metastases revealed that ADAM9 is expressed by stromal liver myofibroblasts, particularly those that are localized within the tumor stroma at the invasive front. These results emphasize the importance of tumor-stromal interactions in invasion and suggest that ADAM9-S can be an important determinant in the ability of cancer cells to invade and colonize the liver.

ADAM Proteins↗
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

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

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

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↗
PubMed2004

Effect of overexpression of constitutively active PKCalpha on rat lacrimal gland protein secretion.

PURPOSE: The lacrimal gland secretes water, electrolytes, and protein into the tear film. Decreased secretion from the lacrimal gland can lead to dry eye syndromes with deleterious effects on vision. Protein kinase C (PKC)-alpha plays a major role in cholinergic- and alpha1-adrenergic-induced protein secretion from the lacrimal gland. This study was undertaken to determine whether activation of PKCalpha alone would induce lacrimal gland protein secretion by examining the effects of overexpression of constitutively active PKCalpha. METHODS: Rat lacrimal gland acini were transduced with an adenovirus containing a gene for constitutively active PKCalpha. Protein secretion was measured in response to cholinergic and alpha1-adrenergic agonist stimulation. RESULTS: More than 84% of acinar cells were transduced, and PKCalpha expression was increased 176-fold. Western blot analysis using an antibody to phosphorylated (activated) PKCalpha indicated that the overexpressed PKCalpha was active, and basal secretion was increased. Cholinergic agonist-stimulated protein secretion was not stimulated above basal secretion, whereas alpha1-adrenergic-agonist-stimulated protein secretion was increased in transduced acini. CONCLUSIONS: Basal lacrimal gland protein secretion can be stimulated by bypassing the release of neurotransmitters and activating PKCalpha, possibly leading to the development of new treatments for dry eye syndromes.

Adenoviridae↗
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↗