A Novel Diacylglycerol-lactone Shows Marked Selectivityin Vitroamong C1 Domains of Protein Kinase C (PKC) Isoforms α and δ as Well as Selectivity for RasGRP Compared with PKCα

Protein kinase C (PKC) family has been an alluring objective for new cancer drug discovery. It has been reported to regulate telomerase in several cancer types. Our team had previously used telomerase to elucidate alpha-fetoprotein (AFP) modulation in hepatocellular carcinoma (HCC). The aim of this study was to investigate the interrelationships among PKC isoforms, telomerase and AFP in HCC. PKCα and PKCδ were the most expressed isoforms in HepG2/C3A, PLC/PRF/5, SNU-387 and SKOV-3 cells. Following the upregulation of AFP using pCMV3-AFP and the human telomerase reverse transcriptase (hTERT) using a construct expressing a wild-type hTERT, and after their inhibition with all-trans retinoic acid and hTERT siRNA each respectively, we found that the expression of PKCα, PKCβI, PKCβII and PKCδ was affected by the variation of AFP and hTERT mRNA levels. An increase in AFP expression and secretion was observed after gene silencing of PKCα, PKCβ, PKCδ, and PKCε in HepG2/C3A. A similar pattern was observed in transfected PLC/PRF/5 cells, however PKCδ isoform silencing decreased AFP expression. Furthermore, telomerase activity was quantified using quantitative telomeric repeat amplification protocol. The variations in hTERT expression and telomerase activity were similar to those of AFP. Further investigation showed that PKC isoforms regulate AFP and hTERT expression levels through PI3K/AKT/mTOR pathway in HepG2/C3A and PLC/PRF/5 cells. Thus, these results show for the first time a possible interrelationship that links PKC isoforms to both AFP and hTERT via PI3K/AKT/mTOR pathway in HCC.

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Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

Journal of Applied Physiology ◽

10.1152/japplphysiol.01248.2003 ◽

2004 ◽

Vol 96 (6) ◽

pp. 2028-2033 ◽

Cited By ~ 15

Author(s):

A. Sundaresan ◽

D. Risin ◽

N. R. Pellis

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Type I Collagen ◽

Human Lymphocytes ◽

Receptor Interaction ◽

Type I ◽

Modeled Microgravity ◽

Calcium Dependent ◽

Kinase C ◽

Pkc Isoforms

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-α, -δ, and -ϵ was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms δ and ϵ were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1- g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cγ in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

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Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin in c-Mpl–Expressing UT-7 Cells

Blood ◽

10.1182/blood.v91.3.813.813_813_822 ◽

1998 ◽

Vol 91 (3) ◽

pp. 813-822 ◽

Cited By ~ 3

Author(s):

Ying Hong ◽

Dominique Dumènil ◽

Bernd van der Loo ◽

Frédérique Goncalves ◽

William Vainchenker ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Subcellular Distribution ◽

Membrane Fraction ◽

Induced Expression ◽

Gm Csf ◽

Kinase C ◽

Pkc Isoforms ◽

Mitogenic Action ◽

Pkc Activation

Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms α and β found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms α and β to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

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Protein kinase C-α isoform is involved in erythropoietin-induced erythroid differentiation of CD34+ progenitor cells from human bone marrow

Blood ◽

10.1182/blood.v95.2.510 ◽

2000 ◽

Vol 95 (2) ◽

pp. 510-518 ◽

Cited By ~ 50

Author(s):

June Helen Myklebust ◽

Erlend B. Smeland ◽

Dag Josefsen ◽

Mouldy Sioud

Keyword(s):

Bone Marrow ◽

Protein Kinase C ◽

Protein Kinase ◽

Progenitor Cells ◽

Erythroid Differentiation ◽

Human Bone ◽

Human Bone Marrow ◽

Kinase C ◽

Pkc Isoforms ◽

Cd34 Cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases involved in many cellular responses. Although the analysis of PKC activity in many systems has provided crucial insights to its biologic function, the precise role of different isoforms on the differentiation of normal hematopoietic progenitor cells into the various lineages remains to be investigated. The authors have assessed the state of activation and protein expression of PKC isoforms after cytokine stimulation of CD34+ progenitor cells from human bone marrow. Freshly isolated CD34+ cells were found to express PKC-, PKC-β2, and PKC-ɛ, whereas PKC-δ, PKC-γ, and PKC-ζ were not detected. Treatment with erythropoietin (EPO) or with EPO and stem cell factor (SCF) induced a predominantly erythroid differentiation of CD34+ cells that was accompanied by the up-regulation of PKC- and PKC-β2 protein levels (11.8- and 2.5-fold, respectively) compared with cells cultured in medium. Stimulation with EPO also resulted in the nuclear translocation of PKC- and PKC-β2 isoforms. Notably, none of the PKC isoforms tested were detectable in CD34+ cells induced to myeloid differentiation by G-CSF and SCF stimulation. The PKC inhibitors staurosporine and calphostin C prevented EPO-induced erythroid differentiation. Down-regulation of the PKC-, PKC-β2, and PKC-ɛ expression by TPA pretreatment, or the down-regulation of PKC- with a specific ribozyme, also inhibited the EPO-induced erythroid differentiation of CD34+ cells. No effect was seen with PKC-β2–specific ribozymes. Taken together, these findings point to a novel role for the PKC- isoform in mediating EPO-induced erythroid differentiation of the CD34+ progenitor cells from human bone marrow.

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Inhibitory Effect of Hypocrellin A on Protein Kinase C in Liver and Skeletal Muscle of Obese Zucker Rats

The American Journal of Chinese Medicine ◽

10.1142/s0192415x03001624 ◽

2003 ◽

Vol 31 (06) ◽

pp. 871-878 ◽

Cited By ~ 6

Author(s):

Xianqin Qu ◽

Lei Dang ◽

J. Paul Seale

Keyword(s):

Skeletal Muscle ◽

Protein Kinase C ◽

Protein Kinase ◽

Inhibitory Effect ◽

Zucker Rats ◽

Dependent Manner ◽

Kinase C ◽

Hypocrellin A ◽

Pkc Isoforms ◽

Obese Zucker Rats

In this ex vivo study, the inhibitory activity of hypocrellin A (HA), a perylene quinonoid pigment isolated from the Chinese medicinal fungus Hypocrella bambuase, on protein kinase C (PKC) enzyme activity in insulin target tissues of obese Zucker rats was assessed. Pre-incubation with HA for 30 minutes significantly inhibited the activity of partially purified PKC enzyme from liver and soleus skeletal muscle in a dose-dependent manner ( IC 50=0.07 and 0.26 μg/ml, respectively). HA produced a greater inhibitory effect in enzyme prepared from the liver than enzyme prepared from soleus muscle. Since total PKC activity in these two insulin target tissues is the net result of several different isoforms of PKC, and PKC-θ is a major isoform expressed in the soleus skeletal muscle, the present data suggest that the naturally occurring compound, HA, may selectively inhibit certain PKC isoforms other than PKC-θ. Further investigations are required to determine which PKC isoforms are most susceptible to HA and whether changes in PKC signaling during treatment with HA can reverse abnormalities of glucose and lipid metabolism in insulin resistant and diabetic states.

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Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

Blood ◽

10.1182/blood-2010-10-312199 ◽

2011 ◽

Vol 118 (2) ◽

pp. 416-424 ◽

Cited By ~ 35

Author(s):

Olga Konopatskaya ◽

Sharon A. Matthews ◽

Matthew T. Harper ◽

Karen Gilio ◽

Judith M. E. M. Cosemans ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Molecular Mechanisms ◽

Thrombus Formation ◽

Dense Granule ◽

Molecular Pathway ◽

Kinase C ◽

Pkc Isoforms ◽

Dense Granule Secretion ◽

Granule Secretion

Abstract Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

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PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells

AJP Renal Physiology ◽

10.1152/ajprenal.1996.271.1.f108 ◽

1996 ◽

Vol 271 (1) ◽

pp. F108-F113 ◽

Cited By ~ 6

Author(s):

M. B. Ganz ◽

B. Saksa ◽

R. Saxena ◽

K. Hawkins ◽

J. R. Sedor

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

De Novo ◽

Mesangial Cells ◽

De Novo Synthesis ◽

Kinase C ◽

Pkc Isoforms ◽

Alpha Beta ◽

Pkc Alpha

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.

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Protein Kinase C Isoform ε Negatively Regulates ADP-Induced Thromboxane Generation by Regulating cPLA2 Activation and Calcium Mobilization In Platelets

Blood ◽

10.1182/blood.v116.21.2020.2020 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2020-2020

Author(s):

Yamini Bynagari ◽

Parth Lakhani ◽

Kamala Bhavaraju ◽

Jianguo Jin ◽

Mario C Rico ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Conflicts Of Interest ◽

Arterial Injury ◽

Calcium Mobilization ◽

Erk Activation ◽

Kinase C ◽

Cpla2 Activation ◽

Pkc Isoforms

Abstract Abstract 2020 Positive regulatory role of Protein Kinase C (PKC) isoforms in platelets have been extensively studied. However, negative regulatory roles of PKCs in platelets are poorly understood. In this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms involved in this process. Pan PKC inhibition with GF109203X potentiated ADP-induced cPLA2 phosphorylation suggesting that PKCs negatively regulate thromboxane generation by regulating cPLA2 activation. Inhibition of PKCs potentiated ADP-induced ERK activation and intracellular calcium mobilization, two upstream signaling molecules of cPLA2.These data suggest that PKCs negatively regulate thromboxane by regulating ERK activation and calcium mobilization, which inturn regulate cPLA2 activation. Pan-PKC inhibition potentiated ADP-induced, P2Y1 receptor-mediated calcium mobilization in platelets independent of P2Y12-receptor. Pretreatment of platelets with GF109203X, a Pan PKC inhibitor, but not Go-6976, a classical PKC isoform inhibitor, potentiated ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We have previously demonstrated that nPKC η, θ, δ positively regulates agonist-induced thromboxane generation in platelets. Thus, we investigated if the role of nPKC ε in ADP-induced thromboxane generation using PKC ε knockout mice (PKCε KO). ADP-induced thromboxane generation in PKC ε KO murine platelets was ten-fold higher than that of wild type platelets. Furthermore, PKC ε KO mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in Tail bleeding experiments. We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and thereby offers protection against thrombosis. Disclosures: No relevant conflicts of interest to declare.

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HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction

Biochemical Journal ◽

10.1042/bj3560097 ◽

2001 ◽

Vol 356 (1) ◽

pp. 97-103 ◽

Cited By ~ 16

Author(s):

Carles GIL ◽

Imane CHAIB-OUKADOUR ◽

Juan BLASI ◽

José AGUILERA

Keyword(s):

Signal Transduction ◽

Protein Kinase C ◽

Protein Kinase ◽

Tyrosine Phosphorylation ◽

Heavy Chain ◽

Tetanus Toxin ◽

Signal Transduction Pathways ◽

Terminal Portion ◽

Kinase C ◽

Pkc Isoforms

A recent report [Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177–182] describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn2+-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease. In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)γ-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (HC-TeTx) is reported. The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-α, −β, −γ and −δ isoforms exists, whereas PKC-∊ showed a slight decrease in its soluble fraction immunoreactivity. The PKC-∊ isoform showed no consistent response. Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCγ-1 and ERK-1/2. The effects shown by the HC-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF. The trkA and ERK-1/2 activation were corroborated using phospho-specific antibodies against trkA phosphorylated on Tyr490, and antibodies against Thr/Tyr phosphorylated ERK-1/2. Moreover, PLCγ-1 phosphorylation was supported by its HC-TeTx-induced translocation to the membranous compartment, an event related to PLCγ-1 activation. Since HC-TeTx is the domain responsible for membrane binding and lacks catalytic activity, the activations described here must be exclusively triggered by the interaction of TeTx with a membrane component.

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