Okadaic acid, a phosphatase inhibitor, decreases macrophage motility

1991 ◽  
Vol 260 (2) ◽  
pp. L105-L112 ◽  
Author(s):  
A. K. Wilson ◽  
A. Takai ◽  
J. C. Ruegg ◽  
P. de Lanerolle
Keyword(s):  
Protein Phosphorylation ◽  
Okadaic Acid ◽  
Light Chain ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Phosphoprotein Phosphatase ◽  
Phosphatase Inhibitor ◽  
Signaling Mechanisms ◽  
Dose Dependent

Cellular locomotion results from a series of spatially and temporally integrated reactions. The coordinated regulation of these reactions requires sensitive intracellular signaling mechanisms. Because protein phosphorylation reactions represent important signaling mechanisms in mammalian cells, we investigated the effect of okadaic acid, a phosphoprotein phosphatase inhibitor, on protein phosphorylation and macrophage motility. Okadaic acid was applied to rat alveolar macrophages, and motility was quantitated by a directed chemotaxis assay. Okadaic acid inhibits macrophage motility in a dose-dependent fashion; the concentrations for 50 and 100% inhibition were 3 and 25 microM, respectively. Protein phosphorylation studies demonstrated a 2.5-fold increase in total protein phosphorylation in macrophages treated with 25 microM okadaic acid. These experiments also demonstrated a dose-dependent increase in the phosphorylation of the 20-kDa light chain of myosin. Moreover, 25 microM okadaic acid 1) maximally increased myosin light chain phosphorylation by 6.6-fold, 2) raised the level of myosin associated with the cytoskeleton from a basal level of 47.0 to 96.7% of the total myosin, and 3) induced profound morphological changes as visualized by scanning electron microscopy. These data correlate an increase in protein phosphorylation with a decrease in macrophage motility. Furthermore, they suggest that phosphoprotein phosphatase inhibition may prevent motility by uncoupling coordinated processes, such as cytoskeletal reorganization, that are essential for macrophage motility.

The protein phosphatase inhibitor okadaic acid induces morphological changes typical of apoptosis in mammalian cells

1991 ◽  
Vol 195 (1) ◽  
pp. 237-246 ◽  
Author(s):  
Roald Bøe ◽  
Bjørn Tore Gjertsen ◽  
Olav Karsten Vintermyr ◽  
Gunnar Houge ◽  
Michel Lanotte ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Morphological Changes ◽  
Phosphatase Inhibitor ◽  
Protein Phosphatase Inhibitor

Protein phosphorylation is suppressed when wheat embryos are hydrated and remain growth arrested

2004 ◽  
Vol 14 (3) ◽  
pp. 287-296 ◽  
Author(s):  
Ryan L. Wagner ◽  
M.K. Walker-Simmons
Keyword(s):  
Triticum Aestivum ◽  
Protein Phosphorylation ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Phosphatase Inhibitor ◽  
Protein Phosphatase Inhibitor ◽  
Wheat Embryos ◽  
High Degree ◽  
H Protein ◽  
Net Protein

An early difference in net protein phosphorylation activity occurred in dormant and after-ripened wheat (Triticum aestivum L.) grains within the first hour of imbibition at 30°C. Embryos from dry, dormant and after-ripened caryopses exhibited a high degree of protein phosphorylation, particularly of four proteins (68, 54, 51 and 42 kDa). Upon hydration, protein phosphorylation activity in dormant embryos decreased within 1 h and reached minimal phosphorylation activity by 5 h. Protein phosphorylation activity in after-ripened (germinable) embryos was not suppressed and remained high during germination. If the hydrated dormant embryos were dried, protein phosphorylation activity was restored. Application of the protein phosphatase inhibitor, okadaic acid, partially overcame dormancy and slowed the decrease in phosphorylation activity. These results suggested that reduction of protein phosphorylation activity slowed the rate of germination.

scholarly journals Mitotic cytosol inhibits invagination of coated pits in broken mitotic cells.

1991 ◽  
Vol 114 (6) ◽  
pp. 1159-1166 ◽  
Author(s):  
M Pypaert ◽  
D Mundy ◽  
E Souter ◽  
J C Labbé ◽  
G Warren
Keyword(s):  
Liquid Nitrogen ◽  
Okadaic Acid ◽  
Hela Cells ◽  
Mammalian Cells ◽  
A431 Cells ◽  
Coated Pits ◽  
Cdc2 Kinase ◽  
Phosphatase Inhibitor ◽  
Interphase Cells ◽  
Mitotic Cells

Receptor-mediated endocytosis is inhibited during mitosis in mammalian cells and earlier work on A431 cells suggested that one of the sites inhibited was the invagination of coated pits (Pypaert, M., J. M. Lucocq, and G. Warren. 1987. Eur. J. Cell Biol. 45: 23-29). To explore this inhibition further, we have reproduced it in broken HeLa cells. Mitotic or interphase cells were broken by freeze-thawing in liquid nitrogen and warmed in the presence of mitotic or interphase cytosol. Using a morphological assay, we found invagination to be inhibited only when mitotic cells were incubated in mitotic cytosol. This inhibition was reversed by diluting the cytosol during the incubation. Reversal was sensitive to okadaic acid, a potent phosphatase inhibitor, showing that phosphorylation was involved in the inhibition of invagination. This was confirmed using purified cdc2 kinase which alone could partially substitute for mitotic cytosol.

scholarly journals The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells

2000 ◽  
Vol 113 (11) ◽  
pp. 1985-1992 ◽  
Author(s):  
G. Valenti ◽  
G. Procino ◽  
M. Carmosino ◽  
A. Frigeri ◽  
R. Mannucci ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Water Permeability ◽  
Intracellular Signaling ◽  
Collecting Duct ◽  
Water Channel ◽  
Maximal Effect ◽  
Intact Cells ◽  
Cd8 Cells ◽  
Phosphatase Inhibitor ◽  
Water Permeability Coefficient

Phosphorylation by kinases and dephosphorylation by phosphatase markedly affect the biological activity of proteins involved in intracellular signaling. In this study we investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on water permeability properties and on aquaporin2 (AQP2) translocation in AQP2-transfected renal CD8 cells. In CD8 cells both forskolin alone and okadaic acid alone increased the osmotic water permeability coefficient P(f) by about 4- to 5-fold. In intact cells, in vivo phosphorylation studies revealed that forskolin stimulation resulted in a threefold increase in AQP2 phosphorylation. In contrast, okadaic acid treatment promoted only a 60% increase in AQP2 phosphorylation which was abolished when this treatment was performed in the presence of 1 μM H89, a specific protein kinase A (PKA) inhibitor. Nevertheless, in this latter condition, confocal microscopy analysis revealed that AQP2 translocated and fused to the apical membrane. Okadaic acid-induced AQP2 translocation was dose dependent having its maximal effect at a concentration of 1 μM. In conclusion, our results clearly indicate that okadaic acid exerts a full forskolin-like effect independent from AQP2 phosphorylation. Thus AQP2 phosphorylation is not essential for water channel translocation in renal cells, indicating that different pathways might exist leading to AQP2 apical insertion and increase in P(f).

The role of protein kinase C in alpha-adrenergic regulation of NaCl(K) cotransport in human airway epithelial cells

1995 ◽  
Vol 268 (3) ◽  
pp. L414-L423 ◽  
Author(s):  
C. M. Liedtke
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Protein Phosphorylation ◽  
Okadaic Acid ◽  
Intracellular Signaling ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Kinase C ◽  
Human Trachea

alpha 1-Adrenergic (alpha 1-AR) agents stimulate NaCl(K) cotransport and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]-specific phospholipase C in human trachea and nasal polyp epithelial cells. One second messenger generated by PtdIns(4,5)P2 degradation is inositol trisphosphate. We now show that diglycerides (DG) are also generated during alpha 1-AR stimulation. In cells prelabeled with [3H]arachidonic acid, alpha 1-AR agents produced a biphasic DG generation in normal and cystic fibrosis (CF) cells that is blocked by pertussis toxin. The early DG peak closely paralleled PtdIns(4,5)P2 degradation, stimulation of cotransport by phorbol 12-myristate 13-acetate (PMA), and inhibition of cotransport by the protein kinase C (PKC) inhibitor staurosporine. This suggests that cotransporter activation requires PKC-protein phosphorylation. This possibility was tested using the protein phosphatase inhibitor okadaic acid. Okadaic acid elevated bumetanide-sensitive Cl efflux. Staurosporine also blocked > 63% of okadaic-acid-stimulated Cl transport. The late DG peak did not support hormone-stimulated cotransport. The results demonstrate that DGs are a pivotal link between alpha 1-AR stimulation and NaCl(K) cotransport activation with a role for PKC and protein phosphorylation. alpha 1-AR intracellular signaling mechanisms apparently operate normally in CF cells.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

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