Dynamics of phosphorylation and assembly of the high molecular weight neurofilament subunit in NB2a/d1 neuroblastoma.
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)
Ralph Lowell Laboratories, McLean Hospital, Belmont, MA 02178.
T B
TB
Shea
R K
RK
Sihag
R A
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Nixon
D000941:Antigens / Q000032:analysis; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008970:Molecular Weight; D009447:Neuroblastoma / Q000378:metabolism*; D016900:Neurofilament Proteins; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011108:Polymers; D012995:Solubility; D014407:Tumor Cells, Cultured
D000941:Antigens; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D011108:Polymers; C051774:neurofilament protein H; D011092:Polyethylene Glycols