Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca2+mobilization and degranulation in mast cells

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca2+concentrations and oscillatory association of PKCβ–enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca2+mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca2+entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.

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Activation of protein kinase C alters the intracellular distribution and mobility of cardiac Na+ channels

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00817.2010 ◽

2012 ◽

Vol 302 (3) ◽

pp. H782-H789 ◽

Cited By ~ 28

Author(s):

Haifa Hallaq ◽

Dao W. Wang ◽

Jennifer D. Kunic ◽

Alfred L. George ◽

K. Sam Wells ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Fluorescent Protein ◽

Living Cells ◽

Oxidase Inhibitor ◽

Intracellular Distribution ◽

Human Embryonic Kidney Cells ◽

Kinase C ◽

Pkc Activation

Na+ current derived from expression of the cardiac isoform SCN5A is reduced by receptor-mediated or direct activation of protein kinase C (PKC). Previous work has suggested a possible role for loss of Na+ channels at the plasma membrane in this effect, but the results are controversial. In this study, we tested the hypothesis that PKC activation acutely modulates the intracellular distribution of SCN5A channels and that this effect can be visualized in living cells. In human embryonic kidney cells that stably expressed SCN5A with green fluorescent protein (GFP) fused to the channel COOH-terminus (SCN5A-GFP), Na+ currents were suppressed by an exposure to PKC activation. Using confocal microscopy, colocalization of SCN5A-GFP channels with the plasma membrane under control and stimulated conditions was quantified. A separate population of SCN5A channels containing an extracellular epitope was immunolabeled to permit temporally stable labeling of the plasma membrane. Our results demonstrated that Na+ channels were preferentially trafficked away from the plasma membrane by PKC activation, with a major contribution by Ca2+-sensitive or conventional PKC isoforms, whereas stimulation of protein kinase A (PKA) had the opposite effect. Removal of the conserved PKC site Ser1503 or exposure to the NADPH oxidase inhibitor apocynin eliminated the PKC-mediated effect to alter channel trafficking, indicating that both channel phosphorylation and ROS were required. Experiments using fluorescence recovery after photobleaching demonstrated that both PKC and PKA also modified channel mobility in a manner consistent with the dynamics of channel distribution. These results demonstrate that the activation of protein kinases can acutely regulate the intracellular distribution and molecular mobility of cardiac Na+ channels in living cells.

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Binding of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein (MRP) to vesicular phospholipid membranes

Biochemical Journal ◽

10.1042/bj3300005 ◽

1998 ◽

Vol 330 (1) ◽

pp. 5-11 ◽

Cited By ~ 26

Author(s):

Guy VERGÈRES ◽

J. Jeremy RAMSDEN

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Phospholipid Vesicles ◽

Related Protein ◽

Effector Domain ◽

Lower Affinity ◽

Kinase Substrate ◽

Kinase C ◽

Central Effector

The myristoylated alanine-rich C kinase substrate (MARCKS) protein family has two known members, MARCKS itself and MARCKS-related protein (MRP, also called MacMARCKS or F52). They are essential for brain development and are believed to regulate the structure of the actin cytoskeleton at the plasma membrane. Hence membrane binding is central to their function. MARCKS has been quite extensively characterized; MRP much less so. Despite the fact that MRP is only two thirds the size of MARCKS, it has hitherto been assumed that the two proteins have similar properties. Here we make a detailed study, including the effects of myristoylation, lipid composition, calmodulin and phosphorylation of the binding of MRP to phospholipid vesicles. We show that both the N-terminal myristoyl moiety and the central effector domain mediate binding. MRP behaves like MARCKS in the presence of neutral phospholipids. In contrast to MARCKS, however, the incorporation of 20% of negatively-charged phospholipids only marginally increases the affinity of myristoylated MRP. Co-operativity between the myristoyl moiety and the effector domain of MRP is weak and the protein has a significantly lower affinity for these vesicles compared with MARCKS. Furthermore, calmodulin or phosphorylation of the effector domain by the catalytic subunit of protein kinase C do not significantly decrease the binding of myristoylated MRP to negatively-charged phospholipid vesicles. Our results show that the mechanisms regulating the interactions of MARCKS and MRP with phospholipid vesicles are, at least quantitatively, different. In agreement with cellular studies, we therefore propose that MARCKS and MRP have different subcellular localization and, consequently, different functions.

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Phospholipase C and Protein Kinase C-β 2 Mediate Insulin-Like Growth Factor II-Dependent Sphingosine Kinase 1 Activation

Molecular Endocrinology ◽

10.1210/me.2011-0101 ◽

2011 ◽

Vol 25 (12) ◽

pp. 2144-2156 ◽

Cited By ~ 12

Author(s):

Hesham M. El-Shewy ◽

Souzan A. Abdel-Samie ◽

Abdelmohsen M. Al Qalam ◽

Mi-Hye Lee ◽

Kazuyuki Kitatani ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase C ◽

Fluorescent Protein ◽

Sphingosine Kinase ◽

Sphingosine Kinase 1 ◽

Membrane Translocation ◽

Kinase C ◽

Sphingosine 1 Phosphate

Abstract We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

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Distinct Effects of Fatty Acids on Translocation of γ- and ε-Subspecies of Protein Kinase C

The Journal of Cell Biology ◽

10.1083/jcb.143.2.511 ◽

1998 ◽

Vol 143 (2) ◽

pp. 511-521 ◽

Cited By ~ 106

Author(s):

Yasuhito Shirai ◽

Kaori Kashiwagi ◽

Keiko Yagi ◽

Norio Sakai ◽

Naoaki Saito

Keyword(s):

Fatty Acids ◽

Arachidonic Acid ◽

Plasma Membrane ◽

Protein Kinase C ◽

Linoleic Acid ◽

Protein Kinase ◽

Fluorescent Protein ◽

Target Site ◽

Simultaneous Application ◽

Kinase C

Effects of fatty acids on translocation of the γ- and ε-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). γ-PKC–GFP and ε-PKC–GFP predominated in the cytoplasm, but only a small amount of γ-PKC–GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on γ-PKC–GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of ε-PKC–GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of ε-PKC–GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of ε-PKC–GFP than that of γ-PKC–GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of γ-PKC–GFP. Simultaneous application of arachidonic acid (25 μM) and DiC8 (10 μM) elicited a slow, irreversible translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of γ- and ε-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.

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The involvement of protein kinase C and actin filaments in cortical granule exocytosis in the rat

Reproduction ◽

10.1530/rep.1.00424 ◽

2005 ◽

Vol 129 (2) ◽

pp. 161-170 ◽

Cited By ~ 30

Author(s):

E Eliyahu ◽

A Tsaadon ◽

N Shtraizent ◽

R Shalgi

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Cortical Granule ◽

Fluorescence Signal ◽

Filamentous Actin ◽

Granule Exocytosis ◽

Kinase C ◽

Exact Function ◽

Cortical Granule Exocytosis

Mammalian sperm–egg fusion results in cortical granule exocytosis (CGE) and resumption of meiosis. Studies of various exocytotic cells suggest that filamentous actin (F-actin) blocks exocytosis by excluding secretory vesicles from the plasma membrane. However, the exact function of these microfilaments, in mammalian egg CGE, is still elusive. In the present study we investigated the role of actin in the process of CGE, and the possible interaction between actin and protein kinase C (PKC), by using coimmunoprecipitation, immunohistochemistry and confocal microscopy. We identified an interaction between actin and the PKC alpha isoenzyme in non-activated metaphase II (MII) eggs and in eggs activated by phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). F-actin was evenly distributed throughout the egg’s cytosol with a marked concentration at the cortex and at the plasma membrane. A decrease in the fluorescence signal of F-actin, which represents its depolymerization/reorganization, was detected upon fertilization and upon parthenogenetic activation. Exposing the eggs to drugs that cause either polymerization or depolymerization of actin (jasplakinolide (JAS) and cytochalasin D (CD) respectively) did not induce or prevent CGE. However, CD, but not JAS, followed by a low dose of TPA doubled the percentage of eggs undergoing complete CGE, as compared with TPA alone. We further demonstrated that myristoylated alanin-rich C kinase substrate (MARCKS), a protein known to cross-link F-actin in other cell types, is expressed in rat eggs and is colocalized with actin. In view of our results, we suggest that the cytoskeletal cortex is not a mere physical barrier that blocks CGE, but rather a dynamic network that can be maneuvered towards allowing CGE by activated actin-associated proteins and/or by activated PKC.

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Phospholipase A(2) and its products are involved in the purinergic receptor-mediated translocation of protein kinase C in CHO-K1 cells

Journal of Cell Science ◽

10.1242/jcs.113.8.1335 ◽

2000 ◽

Vol 113 (8) ◽

pp. 1335-1343

Author(s):

Y. Shirai ◽

K. Kashiwagi ◽

N. Sakai ◽

N. Saito

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Purinergic Receptor ◽

Phospholipase A ◽

Ionophore A23187 ◽

Dependent Manner ◽

Kinase C ◽

Pkc Epsilon ◽

Phospholipase A 2

The signal transduction involved in the purinergic stimuli-induced activation of protein kinase C (PKC) in CHO-K1 cells was investigated. Purinergic stimuli such as adenosine triphosphate and uridine triphosphate induced a transient translocation of PKC epsilon, gamma, and delta from the cytoplasm to the plasma membrane. These translocations were blocked by an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), but not by an inhibitor of phosphatidylcholine-specific PLC. A diacylglycerol (DAG) analogue also induced reversible translocations of PKC gamma, epsilon, and delta from the cytoplasm to the plasma membrane, while the calcium ionophore A23187 caused a similar translocation of only the gamma subtype. These results confirm that the hydrolysis of phosphatidylinositol-2-phosphate by PLC and the subsequent generation of DAG and increase in Ca(2+)are involved in the purinergic stimuli-induced translocation of PKC. A DAG antagonist, 1-o-hexadecyl-2-o-acetyl-glycerol, blocked the DAG analogue-induced translocations of all PKC subtypes tested but failed to inhibit the purinergic stimuli-induced translocations of PKC epsilon and gamma. The DAG antagonist could not block the ATP- and UTP-induced translocation of PKC epsilon even in the absence of extracellular Ca(2+). Co-application of the DAG antagonist and a phospholipase A(2) (PLA(2)) inhibitor such as aristolochic acid, arachidonyltrifluoromethyl ketone, or bromoenol lactone inhibited the purinergic receptor-mediated translocation of PKC epsilon although each PLA(2) inhibitor alone did not block the translocation. In contrast to the epsilon subtype, ATP-induced translocation of PKC gamma was observed in the presence of both the PLA(2) inhibitor and the DAG antagonist. However, it is noteworthy that re-translocation of PKC gamma was hastened by the PLA(2) inhibitor. Furthermore products of PLA(2), such as lysophospholipids and fatty acids, induced the translocation of PKC gamma and epsilon in a dose dependent manner, but not delta. These results indicate that, in addition to PLC and DAG, PLA(2) and its products are involved in the purinergic stimuli-induced translocation of PKC epsilon and gamma in CHO-K1 cells. Each subtype of PKC in CHO-K1 cell is individually activated in response to a purinergic stimulation.

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Pancreastatin activates protein kinase C by stimulating the formation of 1,2-diacylglycerol in rat hepatocytes

Biochemical Journal ◽

10.1042/bj3030051 ◽

1994 ◽

Vol 303 (1) ◽

pp. 51-54 ◽

Cited By ~ 23

Author(s):

V Sánchez-Margalet ◽

M Lucas ◽

R Goberna

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Pertussis Toxin ◽

Time Course ◽

Rat Hepatocytes ◽

Isolated Hepatocytes ◽

Dependent Manner ◽

Kinase C ◽

Glucose Output ◽

Stimulation Of

We describe here the stimulation by pancreastatin of 1,2-diacylglycerol production and protein kinase C activity in liver plasma membrane and isolated hepatocytes. The dose-dependency for the stimulation of both processes was similar to the recently described pattern of glucose output and cytosolic Ca2+ transients produced by pancreastatin. The time course of diacylglycerol production at 30 degrees C showed a rapid increase within 5 min, reaching a maximum at 10 min. Protein kinase C from hepatocytes was dependent on Ca2+ and phosphatidylserine. Neither the pancreastatin-stimulated diacylglycerol production nor the activation of protein kinase C was affected by pretreatment with pertussis toxin. However, the presence of GTP partially inhibited this pancreastatin stimulation of 1,2-diacylglycerol in a dose-dependent manner, although GTP alone stimulates diacylglycerol accumulation. This inhibitory effect of GTP on pancreastatin stimulation of diacylglycerol synthesis was completely abolished by the pretreatment with pertussis toxin. In conclusion, this study provides evidence that pancreastatin stimulates the formation of 1,2-diacylglycerol by a pertussis-toxin-independent mechanism, which may be responsible for the pancreastatin activation of protein kinase C.

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Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649673 ◽

1993 ◽

Vol 70 (05) ◽

pp. 800-806 ◽

Cited By ~ 29

Author(s):

C Ternisien ◽

M Ramani ◽

V Ollivier ◽

F Khechai ◽

T Vu ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tissue Factor ◽

Factor Ix ◽

Transcriptional Level ◽

Mrna Levels ◽

Dependent Manner ◽

Human Monocytes ◽

Kinase C ◽

Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

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