Methylmalonic acid reduces the in vitro phosphorylation of cytoskeletal proteins in the cerebral cortex of rats.
The present work was undertaken to determine the action of methylmalonic acid (MMA), a metabolite, which accumulates in high amounts in methylmalonic acidemia, on the endogenous phosphorylating system associated with the cytoskeletal fraction proteins of cerebral cortex of young rats. We demonstrated that pre-treatment of cerebral cortex slices of young rats with 2.5 mM buffered methylmalonic acid (MMA) is effective in decreasing in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulins. Based on the fact that this system contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 1 (PP1), we first tested the effect of MMA on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. We observed that MMA totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, the metabolite partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. Furthermore, in vitro dephosphorylation of neurofilament subunits and tubulins was totally inhibited in brain slices pre-treated with MMA. Taken together, these results suggest that MMA, at the same concentrations found in tissues of methylmalonic acidemic children, inhibits the in vitro activities of PKA, CaMKII and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats, a fact that may be involved with the pathogenesis of the neurological dysfunction characteristic of methylmalonic acidemia.
De Mattos-Dutra
A
A
Departamento de Bioquímica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre RS, Brazil.
De Freitas
M S
MS
Schröder
N
N
Zilles
A C
AC
Wajner
M
M
Pessoa-Pureur
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002540:Cerebral Cortex / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D008764:Methylmalonic Acid / Q000494:pharmacology*; D016900:Neurofilament Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism
D003598:Cytoskeletal Proteins; D016900:Neurofilament Proteins; D010761:Phosphorus Radioisotopes; D014404:Tubulin; C044882:neurofilament protein L; C061195:neurofilament protein M; D000255:Adenosine Triphosphate; D008764:Methylmalonic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1