Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo.
The phosphorylation and dephosphorylation of specific proteins was demonstrated directly in the intact vertebrate nervous system in vivo. By exploiting the neurons' ability to segregate a select group of cytoskeletal proteins from most other phosphorylated constituents of the cell by axoplasmic transport, we were able to examine the dynamics of phosphate turnover on neurofilament proteins in mouse retinal ganglion cell neurons simultaneously labeled with [32P]orthophosphate and [3H]proline in vivo. Three [3H]proline-labeled neurofilament protein (NFP) subunits, designated H (160-200 kDa), M (135-145 kDa), and L (68-70 kDa), entered optic axons in a mole:mole ratio similar to that of isolated axonal neurofilaments, supporting the notion that newly synthesized NFPs are transported along axons as assembled neurofilaments. NFP subunits incorporated high levels of 32P before reaching axonal sites at the level of the optic nerve. As neurofilaments were transported along axons, however, many initially incorporated [32P]phosphate groups were removed. Loss of these phosphate groups occurred to a different extent on each subunit. A minimum of 50-60 and 35-40% of the labeled phosphate groups was removed in a 5-day period from the L and M subunits, respectively. By contrast, the H subunit exhibited relatively little or no phosphate turnover during the same period. Dephosphorylation of L in axons is accompanied by a decrease in its net state of phosphorylation; changes in the phosphorylation state of H and M, however, also reflect ongoing addition of phosphates to these polypeptides during axonal transport (Nixon, R.A., Lewis, S.E., and Marotta, C.A. (1986) J. Neurosci., in press). The possibility is raised that dynamic rearrangements of phosphate topography on NFPs represent a mechanism to coordinate interactions of neurofilaments with other proteins as these elements are transported and incorporated into the stationary cytoskeleton along retinal ganglion cell axons.
D000818:Animals; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011392:Proline / Q000378:metabolism; D014316:Tritium
D003598:Cytoskeletal Proteins; D007381:Intermediate Filament Proteins; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D014316:Tritium; D011392:Proline