Co-purification and localization of Munc18-1 (p67) and Cdk5 with neuronal cytoskeletal proteins

Neurochem Int. 2004 Jan;44(1):35-44. doi: 10.1016/s0197-0186(03)00099-8.

Abstract

Munc18-1 (p67, nSec1, rbSec1), a neuron-specific 67kDa protein was independently identified as a syntaxin-binding protein, and as a component that co-purifies with, and regulates the kinase activity of cyclin dependent kinase (Cdk5). Gene knockout studies have demonstrated a role for Munc18-1 in synaptic vesicle docking and neurotransmitter release. Mice lacking Munc18-1 gene were synaptically silent, but the gene deletion did not prevent normal brain assembly, including the formation of layered structures, fiber pathways and morphologically defined synapses. Previous study has shown that Munc18-1 facilitates Cdk5 mediated phosphorylation of KSPXK domains of the neuronal cytoskeletal elements, suggesting that Munc18-1 may function in the regulation of cytoskeletal dynamics. Present study demonstrates the co-purification and co-localization of Munc18 with cytoskeletal elements and forms first step towards understanding the role for Munc18-1 in cytoskeletal dynamics. Conversely, the cytoskeletal proteins and Cdk5 co-purifies with Munc18-1 in a Munc18-1 immuno-affinity chromatography, suggesting a strong protein-protein interaction. Findings from immunofluorescence studies in PC12 cells have shown co-localization of Munc18-1 and Cdk5 with neurofilaments and microtubules. Further, immunohistochemical and immuno-electron microscopic studies of rat olfactory bulb also demonstrated co-localization of Munc18-1 and Cdk5 with cytoskeletal elements. Thus, the biochemical evidence of strong interaction between Munc18-1 with cytoskeletal proteins and morphological evidence of their (Munc18 and cytoskeletal elements) identical sub-cellular localization is suggestive of the possible role for Munc18-1 in cytoskeletal dynamics.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cyclin-Dependent Kinase 5
  • Cyclin-Dependent Kinases / metabolism*
  • Cytoskeletal Proteins / metabolism*
  • Fluorescent Antibody Technique
  • Immunochemistry
  • Immunohistochemistry
  • Male
  • Microscopy, Immunoelectron
  • Munc18 Proteins
  • Nerve Tissue Proteins*
  • Neurons / metabolism*
  • Neurons / ultrastructure
  • Olfactory Bulb / metabolism
  • Olfactory Bulb / ultrastructure
  • PC12 Cells
  • Proteins / chemistry
  • Proteins / metabolism*
  • Rats
  • Rats, Wistar
  • Solubility
  • Vesicular Transport Proteins*

Substances

  • Cytoskeletal Proteins
  • Munc18 Proteins
  • Nerve Tissue Proteins
  • Proteins
  • Stxbp1 protein, rat
  • Vesicular Transport Proteins
  • Cyclin-Dependent Kinase 5
  • Cdk5 protein, rat
  • Cyclin-Dependent Kinases