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ajochen. J. (1997)321. 211—216 (Printed in Greal Br(¡ain) 211
Involvement of Ca2~/caImoduIin-dependent pratein kinase hin the
activation of carnitine palm¡tayltransferase ¡ by okadaic acid in rat
hepatocytes
Guillermo VELASOO*, Manue¡ GUZMÁN*, Victor A. ZAMMITt and Math J. H. GEELENt§
tepartmenl of Bioehemistry and Moiecul& B¡o)ogy 1, Facu)ty of Bio)ogy, Complutense University, 28040-Madrid, Spain, tHannán Research lasfitute, Ayr KA6 SHL,
Scot)and, U.K. aid Laboratory of Veterinary 8)ocremistry aid Instituís of Biomenibranes, U¡echt Univsrsity, 3508 TO Utrechí, lbs Netherlands
The present ‘york was undertaken to study the mechanism by
~vhichokadaic acid (OA). an inhibitor of protein phosphatases 1
and 2A. stimulates carnitine palmitoyltransferase 1 (CPT-1) in
isolated rat hepatocytes [Guzmán. Kolodziej, Caldwell. Costorphine
and Zammit (¡994) Biochem. .1. 300. 693—699]. The OAinduced
srimttlation of CPT-l was aholished bx’ the general
protein kinase inhibitor K-252a a; weli os by KN-62. a specific
inhibitor of Ca24/calmodulin-dependent protein kinase II
(Ca2JCM-PKII). However, neither the protein kinase C-specific
inhibitor bisindolylmaleimide nor the protein kinase A/protein
kinase C inhibitor 1-1-7 was able lo prevent the OA-induced
stimulacion ofCPT-i. Hepatocyte-shrinkage-induced stimulation
INTRODIJCTION
Garnitine pa¡mitoyltransferase 1 (CPT-l) catalyses the pacesetting
step of ¡ong-chain fatty acid transiocation into the
mitochondrial matrix [1—3].It 1; weIl established thac long-taran
changas ini rat liver CPT-l activity occur in response to alterations
ini the nutritional and hormonal status of the animal [1—3].Ini
addition. several studies using permeabilized hepatocvtes haya
.shown that various agents exert short-term effects on CPT-l
activity in parallel with changas ini the rata of Iong-chain fatty
acid oxidation measured in the sama cali praparationis (reviewed
in (3]). ¡ni particular. okadaic acid (OA). a potent inhibitor of
protein phosphatases 1 and 2A [4]. is able to stimuiate by up to
5000 hepatic CPT-I and palmitate oxidation [56], indicating that
inhibition of the phosphatases might have resultad in increased
phosphoryiation of CPT-l, with consaquent increasa in enzyme
acíivity [5.6].However, when hepatocytes were treated with OA
and then permeabilized with digitonin, addition of purified
protein phosphatases 1 and 2A to the permeabilized cali ghosts
did not reverse the OA-induced stimulation of CPT-1 [71. In
addition, although tha effects of OA couid be observad ini
peraneabilized hapatocytes, they were lost upon isolation of
mitochondria from the sama celis. This and other observations
showed that (1) the morcase in CPT-I activity observad in OAtreated
hepatocytes is not due to direct phosphorylation of the
GPT-¡ enzyma. and (Ii) diffusible ccli component(s) iost on
permeabilization of the hepatoc~íe plasmo membrana with
digitonun and distinct from protein phosphatases 1 and 2A are
essenuial for íhe stimulatory effact of OA tobe damonstrated [7].
of CPT-l a; well as OA-induced hepatocyte shrinkage was
prevented by KN-62. KN-62 also antagonized the OA-enhanced
release of lactate dehydrogenase from digitonin-permeabilized
hepatocytes. Exposure of 32P-labelled hepatocytes to OA increased
the degree of phosphorylation of CaC+/CM~PK1I, -os
immunoprecipitated by a monoclonal antibody raised against
the ~-subunit of rat brain kinase. This effect of OA was also
antagonized by KN-62. The resuits thus indicate that the OAdependent
stimulation of CPT-1 may be mediated (at least ini
pan) by increased phosphor~iation anid subsequent activation of
Ca2~/CM-PKli.
The present study was thus conducted to identify intermediate
proteins the phosphoryiation (= activation) of which could be
triggerad by OA, resulting in subsequent activation of CPT-I.
OA may mnifluence the phosphorylation state of a particular
rargat protein either by the direct inhibition of the protein
phosphatases involved in the dephosphorylation of such protein
or by the indirect activation of the protein kiaases involved in its
activation. Regarding the latter possibility, CaC+/calmoduiin~
dapandent protein kinase II (CaJCM-PKII) is abie to become
constitutively activated when autophosphoryiated in key serme
or threonine residues on tha autonomy site of the enzyme [8.9].
Autophosphoryiation is sufficient to disrupt the autoinhibitory
domain of Ca2t/CM-PKII, ieading to a deinhibition of the
kinase [8].1-lance permanent activation of Ca24/CM-PKII should
be achieved by inhibition of the phosphatases involved in the
daphosphoryiation (= deactivation) of Caa±/CM~PKII.la fact.
sevaral responses of intacthepatocytes to OA, namely disruption
of nhe cytoskeieton [10], inhibition of autophagy [10.11] and
activation of phenylaianine hydroxyiase [12], appear to be
mediated by the activation of Ca2~/CM-PKIl. Likewise, in the
present report we prasent data indicating that Ca2~/CM-PKII is
involved in the activation of CPT-I by OA in rat hepatocytes.
EXPERIMENTAL
Materlais
L-(rnetht’1-’4C]Carnitine anid carrier-free [:¡aP]P~were from Amersham
International (Amersham. Bucks., ¡3K.). Phosphate-free
Abbreviations used: Ca2~/CM-PKFI. Ca2tÍcaImodu)in~dependent protein kirtase II: 0PTA, carnitine palmitoyltransferase 1; LDH, lactate
dehydroqenase: CA, okadaic acM.
$ To wbom correspondence shoutd be addressed.