Ca(2+)-mediated phosphorylation and proteolysis activity associated with the cytoskeletal fraction from cerebral cortex of rats.
We describe a Triton-insoluble cytoskeletal fraction extracted from cerebral cortex of young rats retaining an endogenous Ca(2+)-mediated mechanism acting in vitro on Ca2+/calmodulin-dependent protein kinase II (CaM-KII) activity and on phosphorylation and proteolysis of the 150 kDa neurofilament subunit (NF-M), alpha and beta tubulin. Exogenous Ca2+ induced a 70% decrease in the in vitro phosphorylation of the NF-M and tubulins and a 30-50% decrease in the total amount of these proteins. However, when calpastatin was added basal phosphorylation and NF-M and tubulin content were recovered. Furthermore, exogenous Ca2+/calmodulin induced increased in vitro phosphorylation of the cytoskeletal proteins and CaM-KII activity only in the presence of calpastatin, suggesting the presence of Ca(2+)-induced calpain-mediated proteolysis. This fraction could be an interesting model to further studies concerning the in vitro effects of Ca(2+)-mediated protein kinases and proteases associated with the cytoskeletal fraction.
de Freitas
M S
MS
Departamento de Bioquimica, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brasil.
de Mattos-Dutra
A
A
Wannmacher
C M
CM
Pessoa-Pureur
R
R
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002540:Cerebral Cortex / Q000648:ultrastructure*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014404:Tubulin / Q000378:metabolism
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D003598:Cytoskeletal Proteins; D016900:Neurofilament Proteins; D014404:Tubulin; C033668:calpastatin; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010450:Endopeptidases; D002118:Calcium