scholarly journals Cell-free fusion of endocytic vesicles is regulated by phosphorylation.

1992 ◽  
Vol 116 (2) ◽  
pp. 331-338 ◽  
Author(s):  
P G Woodman ◽  
D I Mundy ◽  
P Cohen ◽  
G Warren
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Kinase Inhibitors ◽  
Vesicle Fusion ◽  
Protein Kinase Inhibitors ◽  
Cdc2 Kinase ◽  
Free System ◽  
A Cell ◽  
Candidate Protein ◽  
Endocytic Vesicles

Okadaic acid and microcystin-LR, both potent inhibitors of protein phosphatases (PP), blocked vesicle fusion in a cell-free system. The effect of okadaic acid was reversed by the purified catalytic subunit of PP2A, but not PP1. Inhibition was gradual, required Mg-ATP, and was reduced by protein kinase inhibitors, indicating that it was mediated via protein phosphorylation. A candidate protein kinase would be cdc2 kinase, which normally is active in mitotic extracts and has been shown to inhibit endocytic vesicle fusion (Tuomikoski, T., M.-A. Felix, M. Dorée, and J. Gruenberg. 1989. Nature (Lond.). 342:942-945). However, it would appear that cdc2 kinase is not responsible for inhibition by okadaic acid. When compared to cytosol prepared from mitotic cells, okadaic acid did not increase cdc2 kinase activity sufficiently to account for the inhibition. In addition, inhibition was maintained when cdc2 protein was depleted from cytosol.

scholarly journals Evaluation of Protein Kinase Inhibitors with PLK4 Cross-Over Potential in a Pre-Clinical Model of Cancer

2019 ◽  
Vol 20 (9) ◽  
pp. 2112 ◽  
Author(s):  
Amreena Suri ◽  
Anders W. Bailey ◽  
Maurício T. Tavares ◽  
Hendra Gunosewoyo ◽  
Connor P. Dyer ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Physiochemical Properties ◽  
Clinical Model ◽  
Anti Cancer ◽  
Brain Exposure ◽  
Dividing Cells ◽  
A Cell ◽  
Embryonal Brain

Polo-like kinase 4 (PLK4) is a cell cycle-regulated protein kinase (PK) recruited at the centrosome in dividing cells. Its overexpression triggers centrosome amplification, which is associated with genetic instability and carcinogenesis. In previous work, we established that PLK4 is overexpressed in pediatric embryonal brain tumors (EBT). We also demonstrated that PLK4 inhibition exerted a cytostatic effect in EBT cells. Here, we examined an array of PK inhibitors (CFI-400945, CFI-400437, centrinone, centrinone-B, R-1530, axitinib, KW-2449, and alisertib) for their potential crossover to PLK4 by comparative structural docking and activity inhibition in multiple established embryonal tumor cell lines (MON, BT-12, BT-16, DAOY, D283). Our analyses demonstrated that: (1) CFI-400437 had the greatest impact overall, but similar to CFI-400945, it is not optimal for brain exposure. Also, their phenotypic anti-cancer impact may, in part, be a consequence of the inhibition of Aurora kinases (AURKs). (2) Centrinone and centrinone B are the most selective PLK4 inhibitors but they are the least likely to penetrate the brain. (3) KW-2449, R-1530 and axitinib are the ones predicted to have moderate-to-good brain penetration. In conclusion, a new selective PLK4 inhibitor with favorable physiochemical properties for optimal brain exposure can be beneficial for the treatment of EBT.

scholarly journals Regulation of transferrin receptor recycling by protein phosphorylation

1994 ◽  
Vol 303 (2) ◽  
pp. 647-655 ◽  
Author(s):  
J R Beauchamp ◽  
P G Woodman
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Cell Lines ◽  
Okadaic Acid ◽  
Transferrin Receptor ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Protein Kinase Inhibitors ◽  
Endocytic Pathway ◽  
Transferrin Uptake

The effect of the protein phosphatase inhibitor okadaic acid on transferrin receptor internalization and recycling was examined in HeLa and K562 cells. Okadaic acid inhibited receptor uptake by more than 85% in both cell lines, whereas it affected transferrin recycling to differing degrees: recycling in HeLa cells was inhibited by greater than 90%, compared with only 65% in K562 cells. Okadaic acid also caused a marked redistribution of receptors in each cell line, which was accounted for by the difference in the extent to which transferrin uptake and recycling were inhibited. These effects were most likely mediated by a protein kinase, as they were delayed by 10-15 min and could be suppressed by prior incubation with certain protein kinase inhibitors. In addition, it was found that specific kinase inhibitors affected basal rates of transferrin uptake and recycling, although the extent of these effects differed between cell lines. Together, these results suggest that a complex pattern of protein phosphorylation influences the flux of the endocytic pathway in interphase cells.

scholarly journals Protein kinase-dependent effects of okadaic acid on hepatocytic autophagy and cytoskeletal integrity

1992 ◽  
Vol 284 (3) ◽  
pp. 633-636 ◽  
Author(s):  
I Holen ◽  
P B Gordon ◽  
P O Seglen
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Kinase Inhibitors ◽  
Specific Inhibitor ◽  
Rat Hepatocytes ◽  
Protein Kinase Inhibitors ◽  
Disruptive Effect ◽  
Isolated Rat Hepatocytes ◽  
Calmodulin Antagonist ◽  
General Protein

The protein phosphatase inhibitor okadaic acid suppressed autophagy completely in isolated rat hepatocytes, as measured by the sequestration of electroinjected [3H]raffinose into sedimentable autophagic vacuoles. Okadaic acid was effectively antagonized by the general protein kinase inhibitors K-252a and KT-5926, the calmodulin antagonist W-7, and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK-II). These inhibitors also antagonized a cytoskeleton-disruptive effect of okadaic acid, manifested as the disintegration of cell corpses after breakage of the plasma membrane. CaMK-II, or a closely related enzyme, would thus seem to play a role in the control of autophagy as well as in the control of cytoskeletal organization.

Activation of deoxycytidine kinase by protein kinase inhibitors and okadaic acid in leukemic cells

2004 ◽  
Vol 68 (1) ◽  
pp. 95-103 ◽  
Author(s):  
Caroline Smal ◽  
Sabine Cardoen ◽  
Luc Bertrand ◽  
Anne Delacauw ◽  
Augustin Ferrant ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Leukemic Cells ◽  
Deoxycytidine Kinase

scholarly journals Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors.

1990 ◽  
Vol 265 (36) ◽  
pp. 22255-22261
Author(s):  
J F Geissler ◽  
P Traxler ◽  
U Regenass ◽  
B J Murray ◽  
J L Roesel ◽  
...  
Keyword(s):  
Biological Activity ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Tyrosine Protein Kinase

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

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