Platelet shape change and protein phosphorylation induced by ADP and thrombin are not sensitive to short periods of microgravity

Recent experiments have shown that the stimulation of animal cells in vitro by direct protein kinase C (PKC) activators is significantly reduced under microgravity (micro g). Platelets undergo protein phosphorylation and morphological changes a few seconds after stimulation by agonists such as phorbol esters which activate PKC. Therefore, taking advantage of parabolic plane flight to obtain short periods of microgravity, we studied phosphorylation of myosin light chain (20K), specific PKC-dependent phosphorylation of a 40,000 M(r) protein, pleckstrin (40K) and platelet shape change. SDS-PAGE analysis and electron microscopy were performed on platelets subjected to 20 seconds microgravity as compared to normal gravity (1 g) conditions. These investigations showed that neither Ca(2+)-calmodulin-mediated activation nor the PKC-dependent pathways are inhibited during short periods of microgravity.

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Arachidonic acid stimulates the formation of 1,2-diacylglycerol and phosphatidic acid in human platelets. Degree of phospholipase C activation correlates with protein phosphorylation, platelet shape change, serotonin release, and aggregation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)44408-5 ◽

1983 ◽

Vol 258 (18) ◽

pp. 11236-11242 ◽

Cited By ~ 21

Author(s):

W Siess ◽

F L Siegel ◽

E G Lapetina

Keyword(s):

Arachidonic Acid ◽

Phosphatidic Acid ◽

Protein Phosphorylation ◽

Phospholipase C ◽

Shape Change ◽

Human Platelets ◽

Serotonin Release ◽

Platelet Shape

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Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2983-2990.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2983-2990

Author(s):

J C Lacal ◽

A Cuadrado ◽

J E Jones ◽

R Trotta ◽

D E Burstein ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Neuronal Differentiation ◽

Phorbol Esters ◽

Ras Oncogene ◽

Intact Cells ◽

Pc 12 Cells ◽

Kinase C ◽

Ras P21

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.

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Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

The Journal of Cell Biology ◽

10.1083/jcb.108.2.553 ◽

1989 ◽

Vol 108 (2) ◽

pp. 553-567 ◽

Cited By ~ 39

Author(s):

V Papadopoulos ◽

P F Hall

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Light Chain ◽

Myosin Light Chain ◽

Phorbol Esters ◽

Intact Cells ◽

Adrenal Cells ◽

Isolation And Characterization ◽

Kinase C

The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.

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Lowering pH in blood platelets dissociates myosin phosphorylation from shape change and myosin association with the cytoskeleton.

The Journal of Cell Biology ◽

10.1083/jcb.105.4.1761 ◽

1987 ◽

Vol 105 (4) ◽

pp. 1761-1769 ◽

Cited By ~ 21

Author(s):

V T Nachmias ◽

K Yoshida ◽

M C Glennon

Keyword(s):

Phorbol Esters ◽

Shape Change ◽

Blood Platelets ◽

Chloride Ion ◽

Weak Acids ◽

Kinase C ◽

Internal Ph ◽

Mlc Phosphorylation ◽

Active Interaction ◽

External Ph

Platelet shape change induced by ADP is relatively independent of external pH over the range 6-7. If the chloride ion in the buffer is replaced by weak acids, however, shape change is rapidly and reversibly inhibited as a function of lowered pH (92% at pH 6.0). This inhibition is correlated with lowered internal pH caused by the weak acids, as measured by the 5,5-dimethyloxazolidine 2,4-dione technique. Shape change was 50% inhibited at internal pH 6.4 when 50 mM NaCl was replaced by propionate (PR). When platelets were stimulated with ADP 10-20 s after addition of PR to a final pH of 6 (PR6), both myosin light chain (MLC) phosphorylation and myosin and actin association with the cytoskeleton were reduced in correlation with the inhibition of shape change. But when ADP was added 30 s after PR6, the MLC phosphorylation was essentially the same in PR or in chloride, although shape change and myosin and actin association with the cytoskeleton remained inhibited. This was shown to be due mainly to endogenous phosphorylation of MLC. On return to neutral pH, platelets in PR immediately changed shape and myosin and actin became associated with the cytoskeleton. Two-dimensional tryptic peptides of MLC showed two major spots after PR6 treatment, indicating that both the MLC kinase site and the protein kinase C sites were phosphorylated. The results show that increased internal pH is not required for shape change, although it may affect the rate. In PR6, as after phorbol esters, MLC phosphorylation can be uncoupled from shape change. The association of myosin and actin with the cytoskeleton is closely correlated with shape change, suggesting that shape change requires the active interaction of these contractile proteins.

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Release of superoxide and change in morphology by neutrophils in response to phorbol esters: antagonism by inhibitors of calcium-binding proteins.

The Journal of Cell Biology ◽

10.1083/jcb.101.3.1052 ◽

1985 ◽

Vol 101 (3) ◽

pp. 1052-1058 ◽

Cited By ~ 53

Author(s):

J M Robinson ◽

J A Badwey ◽

M L Karnovsky ◽

M J Karnovsky

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Guinea Pig ◽

Calcium Binding ◽

Binding Proteins ◽

Phorbol Esters ◽

Morphological Changes ◽

Calcium Binding Proteins ◽

Kinase C ◽

Activate Protein Kinase

The ability of phorbol derivatives to function as stimulating agents for superoxide (O2-) release by guinea pig neutrophils has been evaluated and compared to the known ability of each compound to activate protein kinase C. Those that activate the kinase also stimulate O2- release, while those that are inactive with respect to the kinase have no effect on O2- release. The same correlation was observed with respect to the ability of phorbol esters to induce morphological changes in neutrophils, i.e., vesiculation and reduction in granule content. Certain phenothiazines and naphthalene sulfonamides that are known antagonists of calcium-binding proteins blocked both phorbol ester-induced O2- release and morphological changes in these cells.

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Role of protein kinase C in U46619-induced platelet shape change, aggregation and secretion

Thrombosis Research ◽

10.1016/0049-3848(89)90171-0 ◽

1989 ◽

Vol 56 (2) ◽

pp. 299-306 ◽

Cited By ~ 11

Author(s):

Tohru Nakano ◽

Kohji Hanasaki ◽

Hitoshi Arita

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Shape Change ◽

Kinase C ◽

Platelet Shape

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Evidence for Ca2+-independent phosphorylation of human platelet myosin

10.1055/s-0038-1644527 ◽

1987 ◽

Author(s):

J L Daniel ◽

M Rigmaiden

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Myosin Light Chain ◽

Shape Change ◽

Human Platelets ◽

Myosin Phosphorylation ◽

Kinase C ◽

Synthetic Inhibitor ◽

Measurable Change ◽

Platelet Shape

Phosphorylation of platelet myosin is thought to be required for activation of the contractile events occurring during platelet activation. At present the only known mechanism for Onitiating myosin phosphorylation is through a Ca2+-calmodulin-dependent activation of myosin light chain kinase. However, our previous studies using the fluorescent Ca2+-indicator quin2 indicated that both platelet shape change and myosin phosphorylation could be induced in an EGTA-containing media in the absence of a measurable change in cytosolic free Ca2+ concentration (Hallam, Daniel, Kendrick-Jones & Rink. Biochem. J. 232 (1985) 373). In order to confirm this finding, we fyave investigated the regulation of myosin phosphorylation usin^+a preparation of electrically-permeabilized platelets and Ca2+ buffers to control the internal Ca2+ concentration. Fifty percent myosin phosphorylation was obtained at 700 nM Ca2+. When thrombin (5 U/ml) was added to this system, this curve shifted both to the left and upward; 50% myosin phosphorylation was obtained at 400 nM Ca2+.A synthetic inhibitor of protein kinase C, H7, had no effect on myosin phosphorylation in the absence of agonist but did inhibit the thrombin-induced shift to left suggesting that protein kinase C may modulate myosin phosphorylation. We also compared the effects of H7 agonist-induced myosin phosphorylation and shape change in control and an quin2 loaded platelets. Comparable inhibition of both phosphorylation and the rate of shape change was observed with both quin2 and H7. Addition of H7 to quin2-loaded platelets resulted in complete inhibition of both agonist-induced shape change and myosin phosphorylation. These results indicate that both protein kinase C and Ca2+-dependent reactions are involved in complete expression of myosin phosphorylation in human platelets.

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Ultrastructural Characterization of Platelet-Activating Factor-Stimulated Human Eosinophils from Patients with Asthma

Clinical Science ◽

10.1042/cs0840391 ◽

1993 ◽

Vol 84 (4) ◽

pp. 391-399 ◽

Cited By ~ 7

Author(s):

Claus Kroegel ◽

Ann Dewar ◽

Tatsuo Yukawa ◽

Per Venge ◽

Peter J. Barnes ◽

...

Keyword(s):

Morphometric Analysis ◽

Morphological Changes ◽

Shape Change ◽

Platelet Activating Factor ◽

Eosinophil Cationic Protein ◽

Cell Periphery ◽

Cationic Protein ◽

Asthmatic Patients ◽

Transmission Electron

1. Purified human eosinophils from asthmatic patients were stimulated with platelet-activating factor in vitro and examined for morphological changes by transmission electron and light microscopy. Changes were also evaluated by morphometric analysis and were related to the platelet-activating factor-stimulated release of granular eosinophil cationic protein. 2. Stimulation of eosinophils with platelet-activating factor induced a dose-dependent shape change, including the elongation of cells, loss of microvilli and the formation of lamellipodia. This effect was maximal at 25 min and was reversible. 3. Stimulation with platelet-activating factor also induced granule movement to the cell periphery and fusion of adjacent granules. Granules became swollen and vesiculated, whereas both the matrix and core showed evidence of solubilization. 4. There was a time-dependent secretion of eosinophilic cationic protein from human eosinophils upon stimulation with platelet-activating factor which occurred without significant lactate dehydrogenase release. 5. Morphometric analysis of the transmission electron micrographs indicated a significant reduction in cytoplasmic area after 10 min of incubation with platelet-activating factor from 39.0 ± 1.7 μm2 for untreated eosinophils to 33.2 ± 2.3 μm2 (P < 0.02) for platelet-activating factor-treated cells, underscoring the observation that the cells change from spherical to ellipsoidal. No significant increase in the perimeter of the cells was found. 6. The number of granule-profiles in platelet-activating factor-stimulated eosinophils was slightly reduced when compared with control, and an increase in granule area was observed 10 min after platelet-activating factor challenge (0.215 ± 0.011 μm2 versus 0.246 ± 0.016 μm2). 7. Human eosinophils from patients with asthma stimulated with platelet-activating factor undergo both cellular and granular alterations and reorganization which parallel the release of granular eosinophil basic protein.

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Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of β2-chimaerin, a ‘non-protein kinase C’ phorbol ester receptor

Biochemical Journal ◽

10.1042/bj20030727 ◽

2003 ◽

Vol 375 (2) ◽

pp. 313-321 ◽

Cited By ~ 71

Author(s):

Maria Jose CALOCA ◽

HongBin WANG ◽

Marcelo G. KAZANIETZ

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phorbol Ester ◽

Phorbol Esters ◽

Gtpase Activating Protein ◽

Protein Activity ◽

Rac Gtpase ◽

Kinase C ◽

Phorbol Ester Receptor

The regulation and function of β2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to β2-chimaerin with high affinity and promote its subcellular distribution. β2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. β2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the β2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of β2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor β2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.

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