Sihag R K, Nixon R A
Laboratory for Molecular Neuroscience, McLean Hospital, Belmont, Massachusetts 02178.
J Biol Chem. 1991 Oct 5;266(28):18861-7.
The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha-Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A-mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport.
70 kDa神经丝蛋白亚基（NF-L）在体内至少三个位点（L1至L3）发生磷酸化（西哈格，R.K.和尼克松，R.A.（1989年）《生物化学杂志》264，457 - 464）。通过向小鼠玻璃体内注射[32P]正磷酸盐使视网膜神经节细胞中的神经丝在体内磷酸化后，测定了轴突运输过程中NF-L上磷酸基团的周转情况。注射后10至90小时的小鼠视神经轴突中NF-L的二维磷酸肽图谱显示，随着神经丝的运输，来自肽L2和L3的放射性标记比来自L1的下降得更快。为了确定肽L2上的磷酸化位点，在肝素存在下，轴突细胞骨架由蛋白激酶A进行磷酸化。分离的NF-L亚基用α-胰凝乳蛋白酶消化后，32P - 肽在反相C8柱上通过高效液相色谱分离。二维肽图谱显示，从蛋白激酶A接受大部分磷酸盐的α-胰凝乳蛋白酶32P - 肽与体内标记的磷酸肽L2迁移情况相同。通过自动埃德曼降解分析该肽的序列（S - V - R - R - S - Y）对应于NF-L序列的氨基酸残基51 - 56。与NF-L的氨基酸残基49 - 61对应的合成13聚体（S - L - S - V - R - R - S - Y - S - S - S - S - G）也被蛋白激酶A磷酸化。13聚体的α-胰凝乳蛋白酶消化产生了一个包含大部分磷酸盐且在二维磷酸肽图谱上与磷酸肽L2共迁移的肽。对磷酸化的13聚体进行埃德曼降解确定丝氨酸残基55作为主要磷酸化位点，该残基位于蛋白激酶A的共有磷酸化序列内。由于蛋白激酶A介导的磷酸化在体外影响中间丝的组装/拆卸，我们提出磷酸肽L2区域是一个神经丝组装结构域，并且在体内亚基合成后相对较早发生的NF-L上Ser-55的磷酸化和去磷酸化循环调节神经丝组装或轴突运输过程中初始相互作用的一个步骤。
