In vitro phosphorylation of cytoskeletal proteins from cerebral cortex of rats

Cláudia Funchal, Lúcia Maria Vieira De Almeida, Samanta Oliveira Loureiro, Lilian Vivian, Priscila De Lima Pelaez, Franciele Dall Bello Pessutto, Aline Meyer Rosa, Moacir Wajner, Regina Pessoa Pureur*

*Corresponding author for this work

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

Procedures for the preparation of high- and low-salt Triton insoluble cytoskeletal fractions from rat brain suitable for studying in vitro phosphorylation by endogenous kinases and phosphatases are described. The high-salt Triton insoluble cytoskeletal fraction is enriched in neurofilament subunits (NF-H, NF-M and NF-L), vimentin and glial fibrillary acidic protein (GFAP), while the low-salt Triton insoluble cytoskeletal fraction contains detergent insoluble cytoskeletal elements such as intermediate filament subunits and tubulins. One of our approaches is to incubate cerebral cortex slices with [32P]orthophosphate before the cytoskeletal fraction extraction, which allows the in vitro phosphorylation of cytoskeletal constituents in an intact intracellular environment. On the other hand, we also incubate low- or high-salt cytoskeletal fractions previously prepared with [γ32P]ATP. By doing so, we are able to study the direct effects of substances on the kinase and phosphatase activities associated with the cytoskeletal fraction. Moreover by using specific activators or inhibitors of protein kinases and phosphatases we can obtain more detailed information on the alterations provoked by these substances. These approaches are useful for the investigation of the neurotoxic effects of various drugs and metabolites affecting the cytoskeletal-associated phosphorylation system in the brain.

Original languageEnglish (US)
Pages (from-to)111-118
Number of pages8
JournalBrain Research Protocols
Volume11
Issue number2
DOIs
StatePublished - May 2003

Keywords

  • Cerebral cortex
  • Cytoskeleton
  • Protein phosphorylation

ASJC Scopus subject areas

  • Neuroscience(all)

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