Malonyl-CoA-independent acute control of hepatic carnitine palmitoyltransferase I activity. Role of Ca2+/calmodulin-dependent protein kinase II and cytoskeletal components.
The mechanism of malonyl-CoA-independent acute control of hepatic carnitine palmitoyltransferase I (CPT-I) activity was investigated. In a first series of experiments, the possible involvement of the cytoskeleton in the control of CPT-I activity was studied. The results of these investigations can be summarized as follows. (i) Very mild treatment of permeabilized hepatocytes with trypsin produced around 50% stimulation of CPT-I activity. This effect was absent in cells that had been pretreated with okadaic acid (OA) and seemed to be due to the action of trypsin on cell component(s) distinct from CPT-I. (ii) Incubation of intact hepatocytes with 3, 3'-iminodipropionitrile, a disruptor of intermediate filaments, increased CPT-I activity in a non-additive manner with respect to OA. Taxol, a stabilizer of the cytoskeleton, prevented the OA- and 3, 3'-iminodipropionitrile-induced stimulation of CPT-I. (iii) CPT-I activity in isolated mitochondria was depressed in a dose-dependent fashion by the addition of a total cytoskeleton fraction and a cytokeratin-enriched cytoskeletal fraction, the latter being 3 times more potent than the former. In a second series of experiments, the possible link between Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII) and the cytoskeleton was studied in the context of CPT-I regulation. The data of these experiments indicate that (i) purified Ca2+/CM-PKII activated CPT-I in permeabilized hepatocytes but not in isolated mitochondria, (ii) purified Ca2+/CM-PKII abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytokeratin-enriched fraction, and (iii) the Ca2+/CM-PKII inhibitor KN-62 prevented the OA-induced phosphorylation of cytokeratins in intact hepatocytes. Results thus support a novel mechanism of short-term control of hepatic CPT-I activity which may rely on the cascade Ca2+/CM-PKII activation --> cytokeratin phosphorylation --> CPT-I de-inhibition.
Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain.
G
G
Velasco
M J
MJ
Geelen
T
T
Gómez del Pulgar
M
M
Guzmán
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D007633:Keratins / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008297:Male; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D014357:Trypsin / Q000378:metabolism
D003598:Cytoskeletal Proteins; D008316:Malonyl Coenzyme A; D007633:Keratins; D002334:Carnitine O-Palmitoyltransferase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin