Dynamics of phosphorylation and assembly of the high molecular weight neurofilament subunit in NB2a/d1 neuroblastoma

J Neurochem. 1990 Nov;55(5):1784-92. doi: 10.1111/j.1471-4159.1990.tb04969.x.


In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton-insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/d1 neuroblastoma cells also contain Triton-soluble, extensively phosphorylated 200-kDa high molecular weight neurofilament subunits (NF-H). High-speed centrifugation (100,000 g) of the Triton-soluble fraction for 1 h sedimented some, but not all, soluble NF-H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF-H subunits in this fraction are assembled into (Triton-soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton-soluble 200-kDa NF-H variants. Because only extensively phosphorylated NF-H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse-chase analyses, radiolabeled 200-kDa NF-H migrated into the 100,000 g particulate fraction of Triton-soluble extracts before its arrival in the Triton-insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton-insoluble, extensively phosphorylated NF-H, contain a sizeable pool of Triton-soluble extensively phosphorylated NF-H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF-H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF-H into neurofilaments and integration into the Triton-insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

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

MeSH terms

  • Antigens / analysis
  • Intermediate Filament Proteins / chemistry
  • Intermediate Filament Proteins / immunology
  • Intermediate Filament Proteins / metabolism*
  • Molecular Weight
  • Neuroblastoma / metabolism*
  • Neurofilament Proteins*
  • Phosphorylation
  • Polyethylene Glycols
  • Polymers
  • Solubility
  • Tumor Cells, Cultured


  • Antigens
  • Intermediate Filament Proteins
  • Neurofilament Proteins
  • Polymers
  • neurofilament protein H
  • Polyethylene Glycols