Neurofilament triplet proteins of NB2a/d1 neuroblastoma: posttranslational modification and incorporation into the cytoskeleton during differentiation.
Induction of axonal neuritogenesis in NB2a/d1 cells was associated with an increased content of neurofilament proteins (NFPs) by immunoblot analysis. The major NFP subunits in differentiated [NB2a(+)] cells included microheterogenous forms with apparent molecular weights of 200-190 kDa (NFP-H), 143-142 kDa (NFP-M) and 70 kDa (NFP-L) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only NFP-L was detected in cytoskeletal preparations of undifferentiated [NB2a(-)] cells. All three NFPs of NB2a(+) cells incorporated 32P-orthophosphate in intact cells. A 160/155 kDa NFP-H immunoreactive polypeptide in NB2a(-) and NB2a(+) cells represented a relatively unmodified form of the 200 kDa NFP-H, since dephosphorylation of the 200 kDa NFP-H in vitro with alkaline phosphatase generated the 160/155 kDa forms. Triton-extracted NB2a(+) cells displayed NFP-H immunoreactivity in neurites and occasionally in perikaryal regions at the base of neurites. NFP-M was present throughout the neurites and somata of NB2a(+) cells, and was regularly detected in portions of perikarya in NB2a(-) cells. NFP-L immunoreactivity was distributed throughout the Triton-insoluble cytoskeleton of NB2a(-) and NB2a(+) cells. Immunocytochemical analyses revealed that extensively phosphorylated forms of NFP-H were largely restricted to the neurites of NB2a(+) cells, and less modified forms predominated throughout both perikarya and neurites of NB2a(-) and NB2a(+) cells.
Shea
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Ralph Lowell Laboratories, Mailman Research Center McLean Hospital, Belmont, MA 02178.
Sihag
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RK
Nixon
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D000818:Animals; D000911:Antibodies, Monoclonal; D001369:Axons / Q000648:ultrastructure; D002454:Cell Differentiation; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D007124:Immunoenzyme Techniques; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000235:genetics*; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D016900:Neurofilament Proteins; D011499:Protein Processing, Post-Translational
D000911:Antibodies, Monoclonal; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins