Naringin-sensitive phosphorylation of plectin, a cytoskeletal cross-linking protein, in isolated rat hepatocytes.
To identify phosphoproteins that might play a role in naringin-sensitive hepatocellular cytoskeletal disruption and apoptosis induced by algal toxins, hepatocyte extracts were separated by gel electrophoresis and immunostained with a phosphothreonine-directed antibody. Use of dilute (5%) polyacrylamide gels containing 6 m urea allowed the resolution of one very large (approximately 500-kDa) okadaic acid- and naringin-sensitive phosphoprotein, identified by tryptic fingerprinting, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and immunostaining as the cytolinker protein, plectin. The naringin-sensitive phosphorylation induced by okadaic acid and microcystin-LR probably reflected inhibition of a type 2A protein phosphatase, whereas the naringin-resistant phosphorylation induced by calyculin A, tautomycin, and cantharidin probably involved a type 1 phosphatase. Okadaic acid caused a collapse of the plectin-immunostaining bile canalicular sheaths and the general cytoskeletal plectin network into numerous medium-sized plectin aggregates. Inhibitors of protein kinase C, cAMP-dependent protein kinase, or Ca(2+)/calmodulin-dependent kinase II had moderate or no protective effects on plectin network disruption, whereas naringin offered 86% protection. Okadaic acid induced a naringin-sensitive phosphorylation of AMP-activated protein kinase (AMPK), the stress-activated protein kinases SEK1 and JNK, and S6 kinase. The AMPK-activating kinase (AMPKK) is likely to be the target of inhibition by naringin, the other kinases serving as downstream components of an AMPKK-initiated signaling pathway.
Larsen
Ann-Kristin Ruud
AK
Proteomics and Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Møller
Michael T N
MT
Blankson
Henrietta
H
Samari
Hamid R
HR
Holden
Lise
L
Seglen
Per O
PO
D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D044950:Flavanones; D005419:Flavonoids / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010449:Peptide Mapping; D010766:Phosphorylation; D051190:Plectin; D047428:Protein Kinase Inhibitors; D051381:Rats; D017208:Rats, Wistar; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004791:Enzyme Inhibitors; D044950:Flavanones; D005419:Flavonoids; D007381:Intermediate Filament Proteins; C495334:Plec protein, rat; D051190:Plectin; D047428:Protein Kinase Inhibitors; D019319:Okadaic Acid; C005274:naringin