Interplay between Calcium, Diacylglycerol, and Phosphorylation in the Spatial and Temporal Regulation of PKCα-GFP

Tanimura, Akihiko; Nezu, Akihiro; Mitsui, Takao; Hashimoto, Noboru; Tojyo, Yosuke

doi:10.1074/jbc.m201130200

Cited by 39 publications

(39 citation statements)

References 31 publications

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“…It is interesting to note that the protein translocation profile obtained after the addition of ionomycin to the medium containing 10 g/ml DiC8 was very similar to that obtained when the cells were stimulated previously with 40 ng/ml antigen (Fig. 2C), suggesting that the concentration of diacylglycerol generated under physiological conditions might also be similar but probably only sufficient to anchor the protein to the plasma membrane for a very short length of time through the C1 domain, as has been suggested in previous reports (8,26).…”

Section: The Ca 2ϩ -Binding Sites Of the C2 Domain Are Critical For Psupporting

confidence: 60%

Role of the Ca2+/Phosphatidylserine Binding Region of the C2 Domain in the Translocation of Protein Kinase Cα to the Plasma Membrane

Bolsover

Gómez‐Fernández

Corbalán-Garcı́a

2003

Journal of Biological Chemistry

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Signal transduction via protein kinase C (PKC) is closely regulated by its subcellular localization. To map the molecular determinants mediating the C2 domaindependent translocation of PKC␣ to the plasma membrane, full-length native protein and several point mutants in the Ca 2؉ /phosphatidylserine-binding site were tagged with green fluorescent protein and transiently expressed in rat basophilic leukemia cells (RBL-2H3). Substitution of several aspartate residues by asparagine completely abolished Ca 2؉ -dependent membrane targeting of PKC␣. Strikingly, these mutations enabled the mutant proteins to translocate in a diacylglycerol-dependent manner, suggesting that neutralization of charges in the Ca 2؉ binding region enables the C1 domain to bind diacylglycerol. In addition, it was demonstrated that the protein residues involved in direct interactions with acidic phospholipids play differential and pivotal roles in the membrane targeting of the enzyme. These findings provide new information on how the C2 domain-dependent membrane targeting of PKC␣ occurs in the presence of physiological stimuli. Protein kinase C family (PKC)1 is known to control many cellular processes including metabolism regulation, receptor signal transduction, cell growth and differentiation, and hormone and neurotransmitter secretion (1, 2). This family consists of 10 closely related isoenzymes that can be divided into three groups according to the type of activator they need. For example, conventional PKCs (␣, ␤I, ␤II, and ␥) require the full complement of negatively charged phospholipids, Ca 2ϩ , and diacylglycerol or phorbol esters before they are activated. The novel PKCs (␦, ⑀, , and ) on the other hand do not require Ca 2ϩ , whereas the atypical PKCs (, /) do not require diacylglycerol or Ca 2ϩ (3).Conventional and novel PKCs share the property of using two membrane-targeting modules for the sensitive, specific, and reversible regulation of their function. Thus, the C1 and the C2 domains are involved in membrane translocation and subsequent enzyme activation (4). Many studies performed to date suggest a sequential mechanism in the activation of PKC␣ by Ca 2ϩ in which membrane association is followed by increased catalytic activity (5-9). However, it is still not clear whether these domains function in a concerted way or as independent modules.The first step of this process is probably the Ca 2ϩ -dependent contact of the C2 domain with negatively charged phospholipids at the plasma membrane (6, 8, 10 -12). Recent studies on different C2 domains reveal that, despite their sequential and architectural similarity, they are functionally specialized modules that exhibit distinct equilibrium and kinetic behaviors that are probably optimized for different Ca 2ϩ -signaling applications (13)(14). Most of these studies have been performed with isolated C2 domains and, in the case of PKC␣, the precise molecular mechanism driving the above interaction and the consequences for the general functioning of the enzyme are still not well defined.Our pr...

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Section: The Ca 2ϩ -Binding Sites Of the C2 Domain Are Critical For Psupporting

confidence: 60%

Role of the Ca2+/Phosphatidylserine Binding Region of the C2 Domain in the Translocation of Protein Kinase Cα to the Plasma Membrane

Bolsover

Gómez‐Fernández

Corbalán-Garcı́a

2003

Journal of Biological Chemistry

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“…Signaling pathways controlling megakaryocytic differentiation of K562 cells It is currently admitted that the effects of phorbol esters are mediated by conventional PKCs a, b1, b2, and g and new PKCs d, e, y, and Z, whose activation requires a relocation from the cytoplasm to the plasma membrane (Schaefer et al, 2001;Tanimura et al, 2002). We used the latter feature to analyse the implication of new and conventional PKCs during megakaryocytic differentiation.…”

Section: Resultsmentioning

confidence: 99%

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis

et al. 2005

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The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA þ SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMAinduced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.

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“…The cell membrane has been suggested to be a target for radiation Drijver, 1979, Somosy, 2000). Membrane permeabilisation induced by saponin resulted in stable PKCa translocation to the membrane (Tanimura et al, 2002), furthermore it was suggested that PKCa may act as an amplifier of Ca 2 þ oscillation. Calcium drives the watery secretion by opening the basolateral K þ and apical Cl À channels, and the osmotic gradient thus created leads to water movement into the lumen both through transcellular and paracellular processes.…”

Section: Discussionmentioning

confidence: 99%

“…If PKCa activation would be found not to be affected by irradiation, this would imply that the relevant radiation-induced damage occurs downstream from the hydrolysis of PIP 2 . To test this we measured the methacholine-induced translocation of PKCa to the cell membrane in cells from irradiated and unirradiated glands, as this has been shown to correlate well with the activation PKCa (Terzian and Rubin 1993;Feng and Hannun 1998;Oancea and Meyer 1998;Tanimura et al, 2002). Stimulation of intact nonirradiated acinar cells from the parotid gland with 10 À4 MCh revealed that a 2 min incubation time induced the highest level of PKCa translocation to the plasma membrane (data not shown).…”

Section: Receptor Density and Affinitymentioning

confidence: 99%

Defects in muscarinic receptor-coupled signal transduction in isolated parotid gland cells after in vivo irradiation: evidence for a non-DNA target of radiation

et al. 2005

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Radiation-induced dysfunction of normal tissue, an unwanted side effect of radiotherapeutic treatment of cancer, is usually considered to be caused by impaired loss of cell renewal due to sterilisation of stem cells. This implies that the onset of normal tissue damage is usually determined by tissue turnover rate. Salivary glands are a clear exception to this rule: they have slow turnover rates (460 days), yet develop radiation-induced dysfunction within hours to days. We showed that this could not be explained by a hypersensitivity to radiation-induced apoptosis or necrosis of the differentiated cells. In fact, salivary cells are still capable of amylase secretion shortly after irradiation while at the same time water secretion seems specifically and severely impaired. Here, we demonstrate that salivary gland cells isolated after in vivo irradiation are impaired in their ability to mobilise calcium from intracellular stores (Ca 2 þ i ), the driving force for water secretion, after exposure to muscarinic acetylcholine receptor agonists. Using radioligandreceptor-binding assays it is shown that radiation caused no changes in receptor density, receptor affinity nor in receptor-G-protein coupling. However, muscarinic acetylcholine agonist-induced activation of protein kinase C alpha (PKCa), measured as translocation to the plasma membrane, was severely affected in irradiated cells. Also, the phorbol ester PMA could no longer induce PKCa translocation in irradiated cells. Our data hence indicate that irradiation specifically interferes with PKCa association with membranes, leading to impairment of intracellular signalling. To the best of our knowledge, these data for the first time suggest that, the cells' capacity to respond to a receptor agonist is impaired after irradiation.

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Interplay between Calcium, Diacylglycerol, and Phosphorylation in the Spatial and Temporal Regulation of PKCα-GFP

Cited by 39 publications

References 31 publications

Role of the Ca2+/Phosphatidylserine Binding Region of the C2 Domain in the Translocation of Protein Kinase Cα to the Plasma Membrane

Role of the Ca2+/Phosphatidylserine Binding Region of the C2 Domain in the Translocation of Protein Kinase Cα to the Plasma Membrane

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis

Defects in muscarinic receptor-coupled signal transduction in isolated parotid gland cells after in vivo irradiation: evidence for a non-DNA target of radiation

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