Phosphorylation of neurofilament proteins in intact neurons: demonstration of phosphorylation in cell bodies and axons

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Abstract
The principal subunits of neurofilaments (NFs) of immature cultured sympathetic neurons have apparent Mr of 68,000 and 145,000; a 200,000 Mr subunit is also present, but at comparatively low levels. These subunits are referred to as the low (NFL), middle (NFM), and high (NFH) Mr subunits, respectively. We studied the phosphorylation of NFL and NFM in these neurons in order to characterize the NFL and NFM isoforms generated by this important posttranslational modification. NFL resolved into a single spot in 2-dimensional gels, although 2 spots were occasionally observed. NFM typically resolved into 3 variants, termed NFM a, b, and c, in order of increasing mobility, but as many as 6 variants were detected in some gels. NFL and, to a much greater degree, NFM became labeled following incubation of intact neurons with 32P-PO4. Although all 3 major NFM variants became labeled, NFM a was the most heavily labeled, followed by NFM b, and then NFM c. Two observations suggest that the generation of these 3 NFM variants is due to their phosphorylation. First, treatment of NFs with phosphatase prior to analysis reduced NFM to a single spot or band that comigrated with NFM c; NFM a and b were completely eliminated. However, NFM c was not fully dephosphorylated because it still reacted with a monoclonal antibody (mAb) specific for a phosphate-dependent epitope on NFM. Second, NFM was recognized by 4 mAbs to distinctly different phosphorylated epitopes of NFM, which suggested that at least 4 distinct sites on NFM can be phosphorylated in cultured neurons. Explant cultures were used to study the phosphorylation of NFL and NFM in cell bodies and axons. In these cultures, a central cell body mass (CBM) 0.5 mm in diameter contains all of the cell bodies, while peripheral to the CBM is a halo of pure axons that extends for 4–6 mm. These cultures were incubated with 32P-PO4 and CBM and axon regions were analyzed separately. NFL became phosphorylated to a greater extent in the CBM than in axons. NFM also became labeled in the CBM and axons, although the relative labeling of NFM a, b, and c in these regions differed considerably from each other and also from the pattern observed in whole neurons (cell bodies plus neurites, see above).(ABSTRACT TRUNCATED AT 400 WORDS)