Localization of myosin phosphatase target subunit 1 in rat brain and in primary cultures of neuronal cells

Beáta Lontay, Zoltán Serfozo, Pál Gergely, Masaaki Ito, David J. Hartshorne, Ferenc Erdodi

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24 Citations (Scopus)

Abstract

Myosin phosphatase (PP1M) is composed of the δ isoform of the PP1 catalytic subunit (PP1cδ), the myosin phosphatase target subunit (MYPT), and a 20 kDa subunit. Western blots detected higher amounts of the MYPT1 isoform compared to MYPT2 in whole brain extracts. The localization of MYPT1 was studied in rat brain and in primary cell cultures of neurons using specific antibodies. Analysis of lysates of brain regions for MYPT1 and PP1M by Western blots using anti-MYPT1 antibodies and by phosphatase assays with myosin as substrate suggested a ubiquitous distribution. Immunohistochemistry of tissue sections revealed that MYPT1 was distributed in all areas of the brain, with staining observed in many different cell types. Depending on the method used for fixation, the MYPT1 appeared with varying intensity in nuclei, in nucleoli, and in the cytoplasm. In primary hippocampal cultures, MYPT1 was identified by confocal microscopy in the cytoplasm and in the nucleus, whereas a predominantly cytoplasmic localization was found in cochlear nucleus cells. In cultured cells, MYPT1 and PP1cδ colocalized with synaptophysin. PP1M activity was high in synaptosomes isolated from the cerebral cortex, but was relatively low in the postsynaptic densities. The interaction of MYPT1 with synaptophysin and with known partners (Rho-kinase, PP1cδ) in brain extracts was shown by immunoprecipitation with anti-MYPT1. Pull-down assays from synaptosomes, using GST-MYPT1, also confirmed these interactions. In conclusion, the widespread cellular and subcellular localization of MYPT1 implies that PP1M may play an important role in the dephosphorylation of key regulatory proteins in neuronal cells.

Original languageEnglish
Pages (from-to)72-87
Number of pages16
JournalJournal of Comparative Neurology
Volume478
Issue number1
DOIs
Publication statusPublished - Oct 4 2004

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Keywords

  • Immunohistochemistry
  • Myosin phosphatase target subunit (MYPT)
  • Protein phosphatase-1 (PP1)
  • Rho-kinase (ROK)
  • Synaptophysin

ASJC Scopus subject areas

  • Neuroscience(all)

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