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.

scholarly journals Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins

2005 ◽  
Vol 386 (2) ◽  
pp. 237-244 ◽  
Author(s):  
Hamid R. SAMARI ◽  
Michael T. N. MØLLER ◽  
Lise HOLDEN ◽  
Tonje ASMYHR ◽  
Per O. SEGLEN
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Rat Hepatocytes ◽  
Calyculin A ◽  
Β Subunit ◽  
Isolated Rat Hepatocytes ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

Autophagic activity in isolated rat hepatocytes is strongly suppressed by OA (okadaic acid) and other PP (protein phosphatase)-inhibitory toxins as well as by AICAR (5-aminoimidazole-4-carboxamide riboside), a direct activator of AMPK (AMP-activated protein kinase). To investigate whether AMPK is a mediator of the effects of the toxin, a phosphospecific antibody directed against the activation of phosphorylation of the AMPK α (catalytic)-subunit at Thr172 was used to assess the activation status of this enzyme. AICAR as well as all the toxins tested (OA, microcystin-LR, calyculin A, cantharidin and tautomycin) induced strong, dose-dependent AMPKα phosphorylation, correlating with AMPK activity in situ (in intact hepatocytes) as measured by the AMPK-dependent phosphorylation of acetyl-CoA carboxylase at Ser79. All treatments induced the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit, consistent with phosphorylation at several sites other than Thr172. The flavonoid naringin, an effective antagonist of OA-induced autophagy suppression, inhibited the AMPK phosphorylation and mobility shifting induced by AICAR, OA or microcystin, but not the changes induced by calyculin A or cantharidin. AMPK may thus be activated both by a naringin-sensitive and a naringin-resistant mechanism, probably involving the PPs PP2A and PP1 respectively. Neither the Thr172-phosphorylating protein kinase LKB1 nor the Thr172-dephosphorylating PP, PP2C, were mobility-shifted after treatment with toxins or AICAR, whereas a slight mobility shifting of the regulatory AMPK β-subunit was indicated. Immunoblotting with a phosphospecific antibody against pSer108 at the β-subunit revealed a naringin-sensitive phosphorylation induced by OA, microcystin and AICAR and a naringin-resistant phosphorylation induced by calyculin A and cantharidin, suggesting that β-subunit phosphorylation could play a role in AMPK activation. Naringin antagonized the autophagy-suppressive effects of AICAR and OA, but not the autophagy suppression caused by cantharidin, consistent with AMPK-mediated inhibition of autophagy by toxins as well as by AICAR.

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 Novel Protein Kinase Induced during Sporangial Cleavage in the Oomycete Phytophthora infestans

2002 ◽  
Vol 1 (5) ◽  
pp. 687-695 ◽  
Author(s):  
Howard S. Judelson ◽  
Samuel Roberts
Keyword(s):  
Protein Kinase ◽  
Phytophthora Infestans ◽  
Protein Kinases ◽  
Kinase Inhibitors ◽  
De Novo ◽  
Protein Kinase Inhibitors ◽  
Kinase Gene ◽  
Mrna Accumulation ◽  
Calmodulin Antagonist ◽  
Zoospore Release

ABSTRACT A study of the effect of inhibitors on zoospore development in Phytophthora infestans demonstrated the involvement of protein kinases and calcium and led to the discovery of a gene induced during zoosporogenesis that encoded a protein resembling Ca+2- and calmodulin-regulated serine/threonine protein kinases. The calcium channel blocker verapamil and the calmodulin antagonist trifluoroperazine inhibited zoosporogenesis and encystment. The protein kinase inhibitors K-252a and KN-93 inhibited zoospore release, encystment, and cyst germination, and K-252a reduced zoospore viability. In contrast, the inhibitors had minor or no effects on sporangia directly germinating in media. Spurred by these findings, a survey of putative protein kinase genes was performed to identify any that were up-regulated during zoosporogenesis. A kinase-encoding gene was identified for which mRNA accumulation was first detected soon after chilling sporangia in water, conditions that induce sporangial cytoplasm to cleave and release zoospores. The transcript persisted in motile zoospores and in germinated cysts but was not detected in other tissues, including hyphae, hyphae placed in water, or directly germinating sporangia. The structure of the predicted protein was novel, as its C-terminal region, which binds calmodulin in related proteins, was unusually short. Concentrations of actinomycin D previously used in experiments that suggested that de novo transcription was not needed for zoosporogenesis or encystment only partially inhibited transcription of the kinase gene, probably due to poor uptake into sporangia.

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 Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes

2003 ◽  
Vol 373 (2) ◽  
pp. 505-513 ◽  
Author(s):  
Michael T. N. MØLLER ◽  
Hamid R. SAMARI ◽  
Monica FENGSRUD ◽  
Per E. STRØMHAUG ◽  
Anne C. ØSTVOLD ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Limited Region ◽  
Flight Mass Spectrometry ◽  
Methylation Capacity ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
Isolated Rat

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S-adenosylmethionine (AdoMet), into S-adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of 32P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and 32P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet–AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.

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.

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