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Cellular Signalling Dec 2017Vav1/2/3 comprise a protein family with guanyl nucleotide exchange activity for Rho and Rac as well as with motifs conferring adapter activity. Biologically, Vav1 plays...
Vav1/2/3 comprise a protein family with guanyl nucleotide exchange activity for Rho and Rac as well as with motifs conferring adapter activity. Biologically, Vav1 plays a critical role in hematologic cell signaling, whereas Vav2/3 have a wider tissue distribution, but all 3 Vav proteins are implicated in cancer development. A structural feature of Vav1/2/3 is the presence of an atypical C1 domain, which possesses close structural homology to the typical C1 domains of protein kinase C but which fails to bind the second messenger diacylglycerol or the potent analogs, the phorbol esters. Previously, we have shown that five residues in the Vav1 C1 domain are responsible for its lack of phorbol ester binding. Here, we show that the lack of phorbol ester binding of Vav3 has a similar basis. We then explore the consequences of phorbol ester binding to a modified Vav3 in which the C1 domain has been altered to allow phorbol ester binding. We find both disruption of the guanyl nucleotide exchange activity of the modified Vav 3 as well as a shift in localization to the membrane upon phorbol ester treatment. This change in localization is associated with altered interactions with other signaling proteins. The studies provide a first step in assessing the potential for the design of custom C1 domain targeted molecules selective for the atypical C1 domains of Vav family proteins.
Topics: Amino Acid Sequence; Humans; Molecular Targeted Therapy; Neoplasms; Phorbol Esters; Protein Domains; Protein Kinase C; Protein Structure, Tertiary; Proto-Oncogene Proteins c-vav; Signal Transduction
PubMed: 28927664
DOI: 10.1016/j.cellsig.2017.09.008 -
The Journal of Biological Chemistry Jul 2003The TRPV4 calcium-permeable channel was cloned from mouse kidney M-1 cells, and the effect of temperature modulation on channel gating/activation by physical and...
Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways.
The TRPV4 calcium-permeable channel was cloned from mouse kidney M-1 cells, and the effect of temperature modulation on channel gating/activation by physical and chemical signals was evaluated. A TRPV4 cDNA construct with a C-terminal V5 epitope was stably transfected into human embryonic kidney (HEK) 293 and Chinese hamster ovary cells resulting in high levels of expression at the plasma membrane. Channel activation was assessed from changes in calcium influx (fura-2 fluorescence measurements) or whole cell currents (patch clamp analysis). At room temperature (22-24 degrees C), exposure of TRPV4-transfected cells to hypotonic medium (225 mOsm/liter) or a non-protein kinase C (PKC)-activating phorbol ester derivative, 4alpha-phorbol 12,13-decanoate (100 nm), induces modest channel activation, whereas phorbol 12-myristate 13-acetate (100 nm), a PKC-activating phorbol ester, and shear stress (3-20 dyne/cm2) had minimal or no effect on channel activation. In contrast, at elevated temperatures (37 degrees C) the channel was rapidly activated by all stimuli. Inhibition of PKC by calphostin C (50 nm) or staurosporine (500 nm) abolished phorbol 12-myristate 13-acetate-induced activation of the channel without affecting the response to other stimuli. Ruthenium red (1 microm) effectively blocked the channel activity by all stimuli. It is concluded that temperature is a critical modulator of TRPV4 channel gating, leading to activation of the channel by a diverse range of microenvironmental chemical and physical signals utilizing a least two transduction pathways, one PKC-dependent and one PKC-independent. The convergence of multiple signals and transduction pathways on the same channel indicate that the channel functions as a molecular integrator of microenvironmental chemical and physical signals.
Topics: Animals; CHO Cells; Calcium; Cation Transport Proteins; Cell Line; Cloning, Molecular; Cricetinae; Cyclic AMP-Dependent Protein Kinases; Humans; Ion Channel Gating; Ion Channels; Mice; Patch-Clamp Techniques; Phorbol Esters; Recombinant Proteins; Ruthenium Red; Signal Transduction; TRPV Cation Channels; Temperature; Tetradecanoylphorbol Acetate; Transfection
PubMed: 12738791
DOI: 10.1074/jbc.M302517200 -
Environmental Health Perspectives Dec 1993The skin, in particular the epidermis, offers unique opportunities to investigate the induction and control of cellular proliferation and tissue homeostasis both under... (Review)
Review
The skin, in particular the epidermis, offers unique opportunities to investigate the induction and control of cellular proliferation and tissue homeostasis both under in vivo and in vitro conditions. Moreover, it represents one of the most feasible model systems for experimental cancer research. As the primary border of the body, the skin has important protective and defensive functions. A general response to external injury consists of a thickening of the epithelial layer (epidermal hyperplasia) combined with an inflammatory reaction. This hyperplastic transformation of the skin is a critical condition of skin tumor development (i.e., conversion and promotion) and of the wound response. It is believed to be due to a transformation of keratinocytes into an activated state characterized by an increased rate of proliferation and the ability to release a series of growth factors and other cytokines that coordinate the defense reaction (e.g., hyperproliferation, recruitment of leukocytes, activation of the immune system) along auto- and paracrine feedback loops. The initial and probably later phases of this response depend critically on a local release of eicosanoids such as prostaglandins and lipoxygenase-generated factors. A unique reaction seen upon phorbol ester treatment of mouse skin is a strong induction of the enzyme 8-lipoxygenase, which might be involved in skin tumor development by catalyzing the generation of clastogenic metabolites thought to play a role in the conversion stage. Hyperplasia may be considered to be the result of an imbalance between the rates of cell gain and cell loss.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Cytokines; Humans; Hyperplasia; Keratinocytes; Models, Biological; Phorbol Esters; Skin; Skin Neoplasms
PubMed: 8013432
DOI: 10.1289/ehp.93101s595 -
The Journal of Biological Chemistry Jan 1989Apolipoprotein E (apoE) is synthesized by the liver and many peripheral cells. Rat ovarian granulosa cells synthesize and secrete apoE, and this apoE production is...
Apolipoprotein E (apoE) is synthesized by the liver and many peripheral cells. Rat ovarian granulosa cells synthesize and secrete apoE, and this apoE production is increased by agents that increase cellular cAMP. In these studies of granulosa cell apoE synthesis we have examined the effect of agents that stimulate various cell kinases, including protein kinases A, G, and C. The cell content of apoE mRNA was measured simultaneously. Cholera toxin (1.25 micrograms/ml), dibutyryl-cAMP (5 mg/ml), and forskolin (10(-4) M), all of which increase cellular cAMP, stimulate apoE accumulation in the medium 7-10-fold. On the other hand, dibutyryl-cGMP (20 mg/ml) has no effect on apoE synthesis or secretion. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (100 ng/ml), a protein kinase C stimulator, increases apoE accumulation in the medium 8-10-fold, while 4 alpha-phorbol 12,13-didecanoate, the inactive phorbol congener, has no such effect. The cAMP effect on apoE synthesis by granulosa cells is maximal at 48 h, while the phorbol ester effect is maximal at 72-96 h in culture. The data indicate that agents whose effects are mediated by activation of protein kinases A and C, but not G, stimulate granulosa cell apoE production. These effects on the amount of secreted apoE are temporally preceded by increases in the granulosa cell content of apoE messenger RNA. Together, these data suggest that the regulation of apoE production in the rat ovarian granulosa cell could involve transcriptional and post-transcriptional mechanisms.
Topics: Animals; Apolipoproteins E; Bucladesine; Cells, Cultured; Cholera Toxin; Colforsin; Cyclic AMP; Female; Granulosa Cells; Kinetics; Phorbol Esters; Protein Kinases; RNA, Messenger; Rats; Rats, Inbred Strains; Reference Values; Tetradecanoylphorbol Acetate
PubMed: 2536028
DOI: No ID Found -
Oxidative Medicine and Cellular... 2016Epithelial-mesenchymal transition (EMT) plays a pivotal role in the progression of cancer, and some transcription factors including Slug and Snail are known to be...
Epithelial-mesenchymal transition (EMT) plays a pivotal role in the progression of cancer, and some transcription factors including Slug and Snail are known to be involved in EMT processes. It has been well established that the excess production of reactive oxygen species (ROS) and epigenetics such as DNA methylation and histone modifications participate in carcinogenesis; however, the cross talk mechanism among EMT, ROS, and epigenetics remains unclear. In the present study, we demonstrated that the treatment of human breast cancer MCF-7 cells with phorbol ester (TPA), a protein kinase C activator, significantly induced cell proliferation and migration, and these were accompanied by the significant induction of Slug expression. Moreover, the TPA-elicited induction of Slug expression was regulated by histone H3 acetylation and NADPH oxidase (NOX) 2-derived ROS signaling, indicating that ROS and histone acetylation are involved in TPA-elicited EMT processes. We herein determined the cross talk mechanism among EMT, ROS, and histone acetylation, and our results provide an insight into the progression of cancer metastasis.
Topics: Acetylation; Blotting, Western; Breast Neoplasms; Cell Proliferation; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Histones; Humans; MCF-7 Cells; NADPH Oxidases; Phorbol Esters; Protein Kinase C; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Signal Transduction; Snail Family Transcription Factors
PubMed: 27127545
DOI: 10.1155/2016/1284372 -
Molecular and Cellular Biology Jan 2019Phorbol 12-myristate 13-acetate (PMA) promotes skin cancer in rodents. The mutations found in murine tumors are similar to those found in human skin cancers, and PMA...
A Tumor-Promoting Phorbol Ester Causes a Large Increase in APOBEC3A Expression and a Moderate Increase in APOBEC3B Expression in a Normal Human Keratinocyte Cell Line without Increasing Genomic Uracils.
Phorbol 12-myristate 13-acetate (PMA) promotes skin cancer in rodents. The mutations found in murine tumors are similar to those found in human skin cancers, and PMA promotes proliferation of human skin cells. PMA treatment of human keratinocytes increases the synthesis of APOBEC3A, an enzyme that converts cytosines in single-stranded DNA to uracil, and mutations in a variety of human cancers are attributed to APOBEC3A or APOBEC3B expression. We tested here the possibility that induction of APOBEC3A by PMA causes genomic accumulation of uracils that may lead to such mutations. When a human keratinocyte cell line was treated with PMA, both APOBEC3A and APOBEC3B gene expression increased, anti-APOBEC3A/APOBEC3B antibody bound a protein(s) in the nucleus, and nuclear extracts displayed cytosine deamination activity. Surprisingly, there was little increase in genomic uracils in PMA-treated wild-type or uracil repair-defective cells. In contrast, cells transfected with a plasmid expressing APOBEC3A acquired more genomic uracils. Unexpectedly, PMA treatment, but not APOBEC3A plasmid transfection, caused a cessation in cell growth. Hence, a reduction in single-stranded DNA at replication forks may explain the inability of PMA-induced APOBEC3A/APOBEC3B to increase genomic uracils. These results suggest that the proinflammatory PMA is unlikely to promote extensive APOBEC3A/APOBEC3B-mediated cytosine deaminations in human keratinocytes.
Topics: Carcinogens; Cell Line; Cell Transformation, Neoplastic; Cytidine Deaminase; Cytosine; DNA, Single-Stranded; Genomics; Humans; Keratinocytes; Minor Histocompatibility Antigens; Mutagenesis; Neoplasms; Phorbol Esters; Proteins; Uracil
PubMed: 30348839
DOI: 10.1128/MCB.00238-18 -
Chembiochem : a European Journal of... Apr 2018To investigate the cellular distribution of tumor-promoting vs. non-tumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin...
To investigate the cellular distribution of tumor-promoting vs. non-tumor-promoting bryostatin analogues, we synthesized fluorescently labeled variants of two bryostatin derivatives that have previously shown either phorbol ester-like or bryostatin-like biological activity in U937 leukemia cells. These new fluorescent analogues both displayed high affinity for protein kinase C (PKC) binding and retained the basic properties of the parent unlabeled compounds in U937 assays. The fluorescent compounds showed similar patterns of intracellular distribution in cells, however; this argues against an existing hypothesis that various patterns of intracellular distribution are responsible for differences in biological activity. Upon further characterization, the fluorescent compounds revealed a slow rate of cellular uptake; correspondingly, they showed reduced activity for cellular responses that were only transient upon treatment with phorbol ester or bryostatin 1.
Topics: Bryostatins; Fluorescent Dyes; Humans; Phorbol Esters; Protein Binding; Protein Kinase C; U937 Cells
PubMed: 29424951
DOI: 10.1002/cbic.201700655 -
Molecular Biology of the Cell Sep 2005Protein kinase D2 (PKD2) belongs to the PKD family of serine/threonine kinases that is activated by phorbol esters and G protein-coupled receptors (GPCRs). Its... (Comparative Study)
Comparative Study
Protein kinase D2 (PKD2) belongs to the PKD family of serine/threonine kinases that is activated by phorbol esters and G protein-coupled receptors (GPCRs). Its C-terminal regulatory domain comprises two cysteine-rich domains (C1a/C1b) followed by a pleckstrin homology (PH) domain. Here, we examined the role of the regulatory domain in PKD2 phorbol ester binding, catalytic activity, and subcellular localization: The PH domain is a negative regulator of kinase activity. C1a/C1b, in particular C1b, is required for phorbol ester binding and gastrin-stimulated PKD2 activation, but it has no inhibitory effect on the catalytic activity. Gastrin triggers nuclear accumulation of PKD2 in living AGS-B cancer cells. C1a/C1b, not the PH domain, plays a complex role in the regulation of nucleocytoplasmic shuttling: We identified a nuclear localization sequence in the linker region between C1a and C1b and a nuclear export signal in the C1a domain. In conclusion, our results define the critical components of the PKD2 regulatory domain controlling phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling and reveal marked differences to the regulatory properties of this domain in PKD1. These findings could explain functional differences between PKD isoforms and point to a functional role of PKD2 in the nucleus upon activation by GPCRs.
Topics: Catalysis; Cell Line; Cell Line, Tumor; Humans; Karyopherins; Nucleocytoplasmic Transport Proteins; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Protein Kinase D2; Protein Kinases; Protein Sorting Signals; Protein Structure, Tertiary; Protein Transport; Receptors, Cytoplasmic and Nuclear; Stomach Neoplasms; Tritium; Exportin 1 Protein
PubMed: 15975900
DOI: 10.1091/mbc.e05-03-0251 -
Journal of Cell Science Jun 2011The signalling lipid phosphatidic acid (PA) is generated by the hydrolysis of phosphatidylcholine (PC), which is catalysed by phospholipase D (PLD) enzymes. Neutrophils,...
The signalling lipid phosphatidic acid (PA) is generated by the hydrolysis of phosphatidylcholine (PC), which is catalysed by phospholipase D (PLD) enzymes. Neutrophils, important cells of the innate immune system, maintain the body's defence against infection. Previous studies have implicated PLD-generated PA in neutrophil function; these have relied heavily on the use of primary alcohols to act as inhibitors of PA production. The recent development of isoform-selective small molecule inhibitors and the generation of a knockout mouse model provide us with accurate tools to study the role of PLDs in neutrophil responses. We show that PLD1 is a regulator of phorbol-ester-, chemoattractant, adhesion-dependent and Fcγ-receptor-stimulated production of reactive oxygen species (ROS) in neutrophils. Significantly we found that this role of PLD is isoform specific: the absence of PLD2 does not negatively affect these processes. Contrary to expectation, other functions required for an efficient immune response operate effectively in Pld2-deficient neutrophils or when both isoforms are inhibited pharmacologically. We conclude that although PLD1 does have important regulatory roles in neutrophils, the field has been confused by the use of primary alcohols; now that gold standard Pld-knockout mouse models are available, previous work might need to be reassessed.
Topics: Animals; Cell Adhesion; Mice; Mice, Knockout; Neutrophils; Phorbol Esters; Phospholipase D; Reactive Oxygen Species; Receptors, IgG; Signal Transduction; Tetradecanoylphorbol Acetate
PubMed: 21610093
DOI: 10.1242/jcs.082008 -
The Journal of Investigative Dermatology May 1988To extend our previous observation in which the binding of resting allogeneic peripheral blood mononuclear leukocytes (PBML) to recombinant gamma-interferon...
Phorbol ester treatment enhances binding of mononuclear leukocytes to autologous and allogeneic gamma-interferon-treated keratinocytes, which are blocked by anti-LFA-1 monoclonal antibody.
To extend our previous observation in which the binding of resting allogeneic peripheral blood mononuclear leukocytes (PBML) to recombinant gamma-interferon (IFN-gamma)-treated keratinocytes was characterized, we examined the influence of phorbol ester activation of the PBML to both autologous and allogeneic IFN-gamma-treated keratinocytes. The activation of PBML by phorbol esters (5 to 100 ng/ml) for brief periods of time (5 min to 1 h) at 37 degrees C led to an increase in the relative percentage of adherence to IFN-gamma-treated keratinocytes from 15% for non-activated PBML to 30% for phorbol ester-treated PBML. A biologically inert phorbol ester derivative did not enhance the binding reaction. No significant binding of phorbol ester-activated PBML was observed to non-IFN-gamma-treated keratinocytes. Both reduction in temperature to 4 degrees C and preincubation of the phorbol ester-treated PBML with anti-LFA-1 monoclonal antibody, led to complete inhibition of this adherence reaction indicating a role for the LFA-1 molecule in phorbol ester-activated PBML/IFN-gamma-treated keratinocyte reactions. Immunophenotypic analysis of the adherent cell population of the phorbol ester-activated PBML to the IFN-gamma-treated keratinocytes revealed that the predominant adherent cell type was the CD8+ T-cell subset (44%) versus the CD4+ T-cell subset (33%) with 23% monocytes and no binding of B lymphocytes. These results suggest that phorbol ester-activated PBML binds twice greater than resting PBML to IFN-gamma-treated keratinocytes, and this increased adherence may further contribute to homing of activated lymphocytes to the epidermis and mononuclear cell trafficking in the skin of inflammatory dermatoses.
Topics: Antibodies, Monoclonal; Antigens; Antigens, Surface; Cell Adhesion; Epidermal Cells; Epidermis; Humans; Interferon-gamma; Keratins; Leukocytes, Mononuclear; Lymphocyte Function-Associated Antigen-1; Phorbol Esters; Time Factors
PubMed: 2452208
DOI: 10.1111/1523-1747.ep12560903