Velasco G, Geelen M J, Gómez del Pulgar T, Guzmán M
Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain.
J Biol Chem. 1998 Aug 21;273(34):21497-504. doi: 10.1074/jbc.273.34.21497.
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.
研究了丙二酰辅酶A非依赖性急性调控肝脏肉碱棕榈酰转移酶I（CPT-I）活性的机制。在第一组实验中，研究了细胞骨架在CPT-I活性调控中的可能作用。这些研究结果总结如下：（i）用胰蛋白酶对透化肝细胞进行非常温和的处理，可使CPT-I活性提高约50%。在用冈田酸（OA）预处理的细胞中未观察到这种效应，这似乎是由于胰蛋白酶对与CPT-I不同的细胞成分的作用。（ii）用中间丝破坏剂3,3'-亚氨基二丙腈孵育完整肝细胞，相对于OA，以非加和方式增加CPT-I活性。细胞骨架稳定剂紫杉醇可阻止OA和3,3'-亚氨基二丙腈诱导的CPT-I刺激。（iii）添加总细胞骨架组分和富含细胞角蛋白的细胞骨架组分后，分离线粒体中的CPT-I活性以剂量依赖性方式降低，后者的效力是前者的3倍。在第二组实验中，在CPT-I调节的背景下研究了Ca2+/钙调蛋白依赖性蛋白激酶II（Ca2+/CM-PKII）与细胞骨架之间的可能联系。这些实验数据表明：（i）纯化的Ca2+/CM-PKII可激活透化肝细胞中的CPT-I，但不能激活分离线粒体中的CPT-I；（ii）纯化的Ca2+/CM-PKII可消除富含细胞角蛋白的组分对分离线粒体中CPT-I的抑制作用；（iii）Ca2+/CM-PKII抑制剂KN-62可阻止OA诱导的完整肝细胞中细胞角蛋白的磷酸化。因此，结果支持一种新的肝脏CPT-I活性短期调控机制，该机制可能依赖于Ca2+/CM-PKII激活→细胞角蛋白磷酸化→CPT-I去抑制的级联反应。
