c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling.

In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit-ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.

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Expression of Ectodermal Neural Cortex 1 and Its Association with Actin during the Ovulatory Process in the Rat

Endocrinology ◽

10.1210/en.2008-1587 ◽

2009 ◽

Vol 150 (8) ◽

pp. 3800-3806 ◽

Cited By ~ 5

Author(s):

Sun-Gyun Kim ◽

Soo-Jeong Jang ◽

Jaemog Soh ◽

Keesook Lee ◽

Jin-Ki Park ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Granulosa Cells ◽

Actin Polymerization ◽

Actin Binding ◽

High Dose ◽

Filamentous Actin ◽

Kinase C ◽

Ovulatory Process

Ectodermal neural cortex (ENC) 1, a member of the kelch family of genes, is an actin-binding protein and plays a pivotal role in neuronal and adipocyte differentiation. The present study was designed to examine the gonadotropin regulation and action of ENC1 during the ovulatory process in immature rats. The levels of ENC1 mRNA and protein were stimulated by LH/human chorionic gonadotropin (hCG) within 3 h both in vivo and in vitro. In situ hybridization analysis revealed that ENC1 mRNA was localized not only in theca/interstitial cells but also in granulosa cells of preovulatory follicles but not of growing follicles in pregnant mare’s serum gonadotropin/hCG-treated ovaries. LH-induced ENC1 expression was suppressed by a high dose of protein kinase C inhibitor RO 31-8220 (10 μm) but not by low doses of RO 31-8220 (0.1–1.0 μm), suggesting the involvement of atypical protein kinase C. ENC1 was detected in both nucleus and cytoplasm that was increased by LH/hCG treatment. Both biochemical and morphological analysis revealed that LH/hCG treatment increased actin polymerization within 3 h in granulosa cells. Interestingly, ENC1 physically associated with actin and treatment with cytochalasin D, an actin-depolymerizing agent, abolished this association. Confocal microscopy further demonstrated the colocalization of ENC1 with filamentous actin (F-actin). The present study demonstrates that LH/hCG stimulates ENC1 expression and increases F-actin formation in granulosa cells. The present study further shows the physical association of ENC1 and F-actin, implicating the role of ENC1 in cytoskeletal reorganization during the differentiation of granulosa cells.

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Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C.

The Journal of Cell Biology ◽

10.1083/jcb.116.3.695 ◽

1992 ◽

Vol 116 (3) ◽

pp. 695-706 ◽

Cited By ~ 98

Author(s):

G P Downey ◽

C K Chan ◽

P Lea ◽

A Takai ◽

S Grinstein

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Actin Cytoskeleton ◽

Protein Phosphorylation ◽

Neutrophil Activation ◽

Human Neutrophils ◽

Actin Assembly ◽

Membrane Ruffling ◽

Kinase C

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.

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Cytokine production in human intestinal mast cells triggered by Fc∈ receptor cross-linking is dependent on both protein kinase C and MAP kinase

Gastroenterology ◽

10.1016/s0016-5085(01)82469-0 ◽

2001 ◽

Vol 120 (5) ◽

pp. A497-A497

Author(s):

A LORENTZ ◽

I KLOPP ◽

T GEBHARDT ◽

M MANNS ◽

S BISCHOFF

Keyword(s):

Protein Kinase C ◽

Mast Cells ◽

Protein Kinase ◽

Map Kinase ◽

Cytokine Production ◽

Fc Receptor ◽

Cross Linking ◽

Kinase C

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The protein kinase C inhibitor H-7 activates human neutrophils: effect on shape, actin polymerization, fluid pinocytosis and locomotion

Journal of Cell Science ◽

10.1242/jcs.96.1.99 ◽

1990 ◽

Vol 96 (1) ◽

pp. 99-106

Author(s):

H.U. Keller ◽

V. Niggli ◽

A. Zimmermann ◽

R. Portmann

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Actin Polymerization ◽

Kinase Inhibitor ◽

Human Neutrophils ◽

Kinase C ◽

Chemotactic Peptides ◽

Protein Kinase C Inhibitor ◽

Shape Changes ◽

Stimulation Of

The present study demonstrates new properties of H-7. The protein kinase inhibitor H-7 is a potent activator of several neutrophil functions. Stimulation of initially spherical nonmotile neutrophils elicits vigorous shape changes within a few seconds, increases in cytoskeletal actin, altered F-actin distribution, increased adhesiveness and a relatively small increase in pinocytic activity. H-7 has also chemokinetic activities. Depending on the experimental condition, H-7 may elicit or inhibit neutrophil locomotion. It failed to induce chemotaxis. Thus, the response pattern elicited by H-7 is different from that of other leukocyte activators such as chemotactic peptides, PMA or diacylglycerols. The finding that H-7 can elicit shape changes, actin polymerization and pinocytosis suggests that these events can occur without activation of protein kinase C (PKC). PMA-induced shape changes and stimulation of pinocytosis were not inhibited by H-7.

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Phosphatidylinositol transfer proteins and protein kinase C make separate but non-interacting contributions to the phosphorylation state necessary for secretory competence in rat mast cells

Biochemical Journal ◽

10.1042/bj3560287 ◽

2001 ◽

Vol 356 (1) ◽

pp. 287-296 ◽

Cited By ~ 4

Author(s):

Jef A. PINXTEREN ◽

Bastien D. GOMPERTS ◽

Danise ROGERS ◽

Scott E. PHILLIPS ◽

Peter E. R. TATHAM ◽

...

Keyword(s):

Protein Kinase C ◽

Mast Cells ◽

Protein Kinase ◽

Aminoglycoside Antibiotic ◽

Pleckstrin Homology Domain ◽

Kinase C ◽

Streptolysin O ◽

Regulatory Machinery ◽

Phosphatidylinositol Transfer ◽

Phosphatidylinositol Transfer Proteins

Mast cells permeabilized by streptolysin O undergo exocytosis when stimulated with Ca2+ and guanosine 5′-[γ-thio]triphosphate but become progressively refractory to this stimulus if it is delayed. This run-down of responsiveness occurs over a period of 20–30min, during which the cells leak soluble and tethered proteins. We show here that withdrawal of ATP during the process of run-down is strongly inhibitory but that as little as 25μM ATP can extend responsiveness significantly; this effect is maximal at 50μM. When phosphatidylinositol transfer proteins (PITPs) are provided to cells at the time of permeabilization, run-down is retarded. We conclude that in the presence of ATP they convey substrates for phosphorylation that are essential for exocytosis and thus interact with the regulatory machinery. Furthermore, we show that PITPα and PITPβ have additive effects in this mechanism, suggesting that they are not functionally redundant. Alternatively, secretion from run-down cells can be inhibited by the aminoglycoside antibiotic neomycin, which is understood to bind to phosphoinositide headgroups, and by a PH (pleckstrin homology) domain polypeptide that binds phosphoinositides. The apparent displacement of neomycin by exogenous PITPs suggests that these proteins screen essential lipids. Secretion from run-down cells is also inhibited by 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG-C16), an inhibitor of protein kinase C. The lack of synergy between neomycin and AMG-C16 suggests that protein kinase C independently provides a second essential component through protein phosphorylation and that there are two independent phosphorylation pathways necessary for secretion competence.

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